HIV envelope polynucleotides and immunogenic composition

ABSTRACT

Oligonucleotide sequences encoding gp120 polypeptides from breakthrough isolates of vaccine trials using MN-rgp120 and the encoded gp120 polypeptides are provided. Use of the gp120 polypeptides from one or more of the isolates in a subunit vaccine, usually together with MN-rgp120, can provide protection against HIV strains that are sufficiently different from the vaccine strain (e.g.; MN-rgp120) that the vaccine does not confer protection against those strains. Antibodies induced by the polypeptides are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/419,362,filed Oct. 15, 1999, now U.S. Pat. No. 6,585,979 which is a divisionalof application Ser. No. 08/889,841 (now U.S. Pat. No. 6,090,392), filedJul. 8, 1997, which claims the benefit of U.S. Provisional ApplicationNo. 60/069,891 filed Jul. 8, 1996 abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to HIV envelope polypeptides and vaccinescontaining the polypeptides.

2. Description of the Related Art

Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirusidentified as the human immunodeficiency virus (HIV). There have beenintense efforts to develop a vaccine that induces a protective immuneresponse based on induction of antibodies or cellular responses. Recentefforts have used subunit vaccines where an HIV protein, rather thanattenuated or killed virus, is used as the immunogen in the vaccine forsafety reasons. Subunit vaccines generally include gp120, the portion ofthe HIV envelope protein which is on the surface of the virus.

The HIV envelope protein has been extensively described, and the aminoacid and nucleic acid sequences encoding HIV envelope from a number ofHIV strains are known (Myers, G. et al., 1992. Human Retroviruses andAIDS. A compilation and analysis of nucleic acid and amino acidsequences. Los Alamos National Laboratory, Los Alamos, N. Mex.). The HIVenvelope protein is a glycoprotein of about 160 kd (gp160) which isanchored in the membrane bilayer at its carboxyl terminal region. TheN-terminal segment, gp120, protrudes into the aqueous environmentsurrounding the virion and the C-terminal segment, gp41, spans themembrane. Via a host-cell mediated process, gp160 is cleaved to formgp120 and the integral membrane protein gp41. As there is no covalentattachment between gp120 and gp41, free gp120 is sometimes released fromthe surface of virions and infected cells.

The gp120 molecule consists of a polypeptide core of 60,000 daltonswhich is extensively modified by N-linked glycosylation to increase theapparent molecular weight of the molecule to 120,000 daltons. The aminoacid sequence of gp120 contains five relatively conserved domainsinterspersed with five hypervariable domains. The positions of the 18cysteine residues in the gp120 primary sequence, and the positions of 13of the approximately 24 N-linked glycosylation sites in the gp120sequence are common to all gp120 sequences. The hypervariable domainscontain extensive amino acid substitutions, insertions and deletions.Sequence variations in these domains result in up to 30% overallsequence variability between gp120 molecules from the various viralisolates. Despite this variation, all gp120 sequences preserve theability of the virus to bind to the viral receptor CD4 and to interactwith gp41 to induce fusion of the viral and host cell membranes.

gp120 has been the object of intensive investigation as a vaccinecandidate for subunit vaccines, as the viral protein which is mostlikely to be accessible to immune attack. At present, clinical trialsusing gp120 MN strain are underway. However, to date no human vaccinetrial has been of sufficient size to confirm or refute vaccine efficacy.

The development of candidate HIV-1 vaccines is burdened by the lack ofin vivo or in vitro models of HIV-1 infection that accuratelyapproximate the conditions of natural infection in humans. Severalcandidate HIV-1 vaccines [Berman et al.; J. Virol. 7:4464–9 (1992);Haigwood et al.; J. Virol. 66:172–82 (1992); Salmon-Ceron et al.; AIDSRes. and Human Retroviruses 11:1479–86 (1995)] have been described thatelicit broadly cross-reactive antibodies able to neutralize a variety ofdiverse HIV-1 isolates in vitro. However, the relevance of in vitroassays to protective immunity in vivo is uncertain. Although severalvaccines have provided chimpanzees with protection from challenge byhomologous and heterologous strains of HIV-1, protection has not alwayscorrelated with in vitro neutralization assays carried out in T celllines, or in lectin and cytokine activated peripheral blood mononuclearcells (PBMCs) [Berman et al.; Nature 345:622–5 (1990); Bruck et al.;Vaccine 12(12):1141–8 (1994); El-Amad et al.; AIDS 9:1313–22 (1995);Girard et al.; J. Virol. 69:6239–48 (1995); and Fulz et al; Science256:1687–1690 (1992)]. While successful protection of chimpanzees isencouraging and has historically proved to be a reliable indicator ofvaccine efficacy, the conditions of infection in all experimental modelsof HIV-1 infection differ significantly from natural infection inhumans.

Experimental HIV-1 infection in vivo and in vitro both suffer from thelimitation that the in vitro amplification of HIV-1, which is requiredto prepare virus stocks for in vitro or in vivo infectivity experiments,imposes a genetic selection that results in a spectrum of virusquasi-species that differ from the spectrum of variants present in theclinical specimens used to establish the culture [Kusumi et al.; J.Virol. 66:875 (1992); Meyerhans et al.; Cell 58:901–10 (1989)]. Becauseof these uncertainties, and even greater uncertainties related to theamount of virus transmitted, the site and cell type involved in initialreplication, and the kinetics of virus dissemination, the ability ofcurrently available in vitro or in vivo assays to reliably predictvaccine efficacy is questionable.

One of the candidate HIV-1 vaccines that have entered human clinicaltrials is recombinant gp120 prepared in Chinese hamster ovary (CHO)cells from the MN strain of HIV-1 (MN-rgp120) (Berman et al.; J. Virol.7:4464–9 (1992)). To date, approximately 499 adults have participated inPhase 1 and 2 immunogenicity and safety trials of this vaccine. The datacollected thus far suggest that MN-rgp120 is safe, immunogenic, andelicits high titers of neutralizing antibodies in greater than 95% ofindividuals immunized according to a 0, 1, and 6 month immunizationschedule [Belshe et al.; JAMA 272(6):475–80 (1994); McElrath; Seminarsin Cancer Biol. 6:1–11 (1995)]. However, during the course of thesetrials, nine vaccinees who received MN-rgp120 have become infected withHIV-1 through high risk behavior. Small trials, such as these, inpopulations with low rates of infection and minimally sized placebocontrol groups do not have sufficient statistical power to confirm orrefute vaccine efficacy.

However, effective vaccines based on gp120 or another HIV protein forprotection against additional strains of HIV are still being sought toprevent the spread of this disease.

DESCRIPTION OF THE BACKGROUND ART

Recombinant subunit vaccines are described in Berman et al.,PCT/US91/02250 (published as number WO91/15238 on 17 Oct. 1991). Seealso, e.g. Hu et al., Nature 328:721–724 (1987) (vaccinia virus-HIVenvelope recombinant vaccine); Arthur et al., J. Virol. 63(12):5046–5053 (1989) (purified gp120); and Berman et al., Proc. Natl. Acad.Sci. USA 85:5200–5204 (1988) (recombinant envelope glycoprotein gp120).

Numerous sequences for gp120 are known. The sequence of gp120 from theIIIB substrain of HIV-1_(LAI) referred to herein is that determined byMuesing et al., “Nucleic acid structure and expression of the humanAIDS/lymphadenopathy retrovirus,” Nature 313:450–458 (1985). Thesequences of gp120 from the NY-5, Jrcsf, Z6, Z321, and HXB2 strains ofHIV-1 are listed by Myers et al., “Human Retroviruses and AIDS; Acompilation and analysis of nucleic acid and amino acid sequences,” LosAlamos National Laboratory, Los Alamos, N. Mex. (1992). The Thai isolateCM244 is described by McCutchen et al., “Genetic Variants of HIV-1 inThailand,” AIDS Res. And Human Retroviruses 8:1887–1895 (1992). TheMN₁₉₈₄ clone is described by Gurgo et al., “Envelope sequences of twonew United States HIV-1 isolates,” Virol. 164:531–536 (1988). As usedherein, MN, MN-rgp120, the MN clone or isolate refers to MH_(GNE). TheMN_(GNE) amino acid sequence is Sequence ID NO:41.

Each of the above-described references is incorporated herein byreference in its entirety.

SUMMARY OF THE INVENTION

Oligonucleotide sequences encoding gp120 polypeptides from breakthroughisolates of vaccine trials using MN-rgp120 and the encoded gp120polypeptides are provided. Use of the gp120 polypeptides from one ormore of the isolates in a subunit vaccine, usually together withMN-rgp120, can provide protection against HIV strains that aresufficiently different from the vaccine strain (e.g.; MN-rgp120) thatthe vaccine does not confer protection against those strains. Antibodiesinduced by the polypeptides are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1G illustrate the kinetics of antibody response to MN-rgp120 invaccinees infected with HIV-1. Sera were collected at the time pointsindicated and assayed for antibodies reactive with MN-rgp120 (opencircles) or a synthetic peptide derived from the V3 domain of MN-rgp120(closed circles). Arrows indicate dates of injection. Plus signindicates the first time HIV-1 infection was detected. Shaded areaindicates data collected after HIV-1 infection. Data from vaccinee C6 isshown in FIG. 1A; C8 in FIG. 1B; C7, FIG. 1C; C11, FIG. 1D; C10, FIG.1E; C17, FIG. 1F; and C15, FIG. 1G.

FIGS. 2A–2G illustrate the kinetics of CD4 blocking antibody response invaccinees infected with HIV-1. Sera were collected at the time pointsindicated and assayed for antibodies able to block the binding of[¹²⁵I]-labeled MN-rgp120 to cell surface CD4. Arrows indicate dates ofinjection. Plus sign indicates the first time HIV-1 infection wasdetected. Shaded area indicates data collected after HIV-1 infection.Data from vaccinee C6 is shown in FIG. 2A; C8 in FIG. 2B; C7, FIG. 2C;C11, FIG. 2D; C10, FIG. 2E; C17, FIG. 2F; and C15, FIG. 2G.

FIGS. 3A–3J illustrate predicted amino acid sequences of envelopeglycoproteins (gp120) from breakthrough viruses. Proviral DNA sequenceswere amplified by PCR from PBMCs and cloned into the PRK5 expressionplasmid. Two clones from each infected vaccinnee were sequenced fromdouble stranded plasmid DNA. Sequence numbering is with reference to theinitiator methionine residue of gp120. For the purpose of comparison,the sequences shown bein at amino acid 12 of the mature, fullyprocessed, envelope glycoproteins (corresponding to position 41 of thegp120 open reading frame). Shaded areas indicate sequences atneutralizing epitopes, dark boxes indicate polymorphisms thought to beimportant for the binding of virus neutralizing Mabs reactive withMN-rgp120. Conserved (C) regions and variable (V) regions are indicatedabove the sequences. Boxes indicate sequence homologies andpolymorphisms. The sequences of the clones shown (i.e., C6.1–C17.3) arefound in the Sequence Listing in SEQ ID NOs: 2, 5, 8, 10, 12, 16, 19,23, 25, 28, 31, 33, 36, and 39, respectively. The sequence of MN_(GNE)appears in the Sequence Listing as SEQ ID NO: 41.

FIG. 4 illustrates immunoprecipitation of recombinant gp120 preparedfrom breakthrough viruses. Recombinant gp120s from the sevenbreakthrough viruses were prepared by transient transfection of 293scells. Cells were metabolically labeled with ³⁵S methionine and growthconditioned cell culture supernatants were immunoprecipitated withpolyclonal antisera to MN-rgp120. Immunoprecipitates were resolved bySDS-PAGE and visualized by autoradiography. C8 lanes a and b correspondto clones C8.3 and C8.6; C6 lanes a and b correspond to clones C6.1 andC6.5; C7 lanes a and b correspond to clones C7.2 and C7.10; C17 lanes aand b correspond to C17.1 and C17.3; C11 lanes a and b correspond toclones C11.5 and C11.7; C10 lanes a and b correspond to clones C10.5 andC10.7; C15 lanes a and b correspond to clones C15.2 and C15.3.

FIGS. 5A–5D illustrate binding of monoclonal antibodies to recombinantgp120 from breakthrough viruses. Growth-conditioned cell culturesupernatants were collected from 293s cells transiently transfected withplasmids directing the expression of breakthrough virus envelopeglycoproteins. The relative rgp120 concentrations were determined byELISA using MAb 5B6 specific for the HSV-1 glycoprotein D flag epitopeat the amino terminus of all of the rgp120 variants described herein.The resulting rgp120 preparations were captured onto wells of microtiterplates coated with a polyclonal antibody specific for a conservedsequence in the C-terminus of gp120. The binding of virus neutralizingmonoclonal antibodies reactive with gp120 was determined by ELISA. FIG.5A, binding by MAb (5B6) specific for the HSV-1 glycoprotein D flagepitope; FIG. 5B, binding by MAb (1034) against the V3 domain ofMN-rgp120; FIG. 5C binding by MAb (50.1) raised against a syntheticpeptide corresponding to the V3 domain of MN-rgp120; FIG. 5D, binding bya human MAb (15e) known to block the binding of gp120 to CD4.

FIG. 6 depicts the mature envelope glycoprotein (gp120) from the MNclone of the MN strain of HIV-1 (SEQ. ID NO: 41). Hypervariable domainsare indicated in bold, and the V and C regions are indicated (accordingto Modrow et al., J. Virology 61(2):570 (1987). Potential glycosylationsites are marked with a (*).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides gp120 polypeptides from breakthroughisolates of HIV vaccine trials. Novel oligonucleotide sequences encodinggp120 from breakthrough isolates which can be used to express gp120 arealso provided. Use of gp120 polypeptides from one or more of theisolates in a subunit vaccine, usually together with MN-rgp120, canprovide protection against HIV strains that are sufficiently differentfrom the vaccine strain (e.g.; MN-rgp120) that the vaccine does notconfer protection against those strains.

In one embodiment, the vaccine is based on the use of the MN-rgp120polypeptide (Sequence ID NO: 41) and gp120 polypeptides from MN-likeviruses that include neutralizing epitopes that are not present in theinitial vaccine strain, and are sufficiently different from those of thevaccine strain, to have been able to cause HIV-1 infections in MN-rgp120vaccinated individuals (i.e.; to result in breakthrough infections). Useof the initial vaccine strain empirically determines the viruses presentin the population that contain additional neutralizing epitopessufficiently different from those of the vaccine strain to escapeprotection induced by the vaccine strain. Use of an initialrepresentative gp120 polypeptide in a vaccine acts as a sieve so thatviruses that are not effectively protected against by the vaccine strainbreakthrough the vaccine, empirically resulting in determination ofadditional strains in a given geographic region that are not isprotected against by the initial vaccine strain. Use of gp120 from thosebreakthrough isolates complements the vaccine isolate by providingadditional neutralizing epitopes not present in the initial vaccinestrain, therefore creating a more complete vaccine that confersprotection against multiple different virus strains in the region.

Prior HIV-1 vaccine strategies were based on selection of appropriatecandidate vaccine polypeptides based on homology alignment studies.However, since some of the neutralizing epitopes areconformation-dependent and the location of all of these epitopes is notknown, this approach necessarily cannot determine all of theneutralizing epitopes that should be included in a vaccine for aparticular region. In contrast, the present approach uses a selectedrepresentative strain and empirically determines strains that aresufficiently different and therefore breakthrough the barrier ofprotection provided by the initial vaccination program. Those strainscan be included in the vaccine to confer more complete protection fromHIV strains in the region. In addition, those strains can be used aloneto confer protection against the breakthrough virus.

In another embodiment, the invention comprises a vaccine containing afirst HIV gp120 polypeptide sequence and a breakthrough isolate HIVgp120 polypeptide sequence from a vaccinee vaccinated with a vaccineincluding the first HIV gp120 polypeptide sequence, the HIV gp120polypeptide sequences being in a suitable carrier. Fragments of one orboth HIV gp120 polypeptide sequences can be substituted for one or bothof the corresponding HIV gp120 polypeptide sequences.

Preferably, the first gp120 polypeptide sequence contains neutralizingepitopes found in one or more gp120 polypeptides present in isolatesfrom the geographical region where the initial vaccine (i.e., thevaccine that gives rise to the breakthrough isolate) is administered.More preferably, the first gp120 polypeptide sequence contains at leastone of the more common neutralizing epitopes for the region, and mostpreferably the first gp120 polypeptide sequence contains at least one ofthe three most common neutralizing epitopes.

gp120 polypeptide sequences suitable for use as the first gp120polypeptide sequence include gp120 MN, the Thai isolate CM244 sequence(hereinafter “gp120 CM244”), gp120 MN-GNE6 (Sequence ID NOs: 43 and 44;also known in the art as “gp120 GNE6”), and gp120 MIN-GNE8 (Sequence IDNO: 46; also known in the art as “gp120 GNE8”), and the like. gp120 MN,gp120 MN-GNE6, and gp120 MN-GNE8 are especially preferred for use as thefirst gp120 polypeptide sequence in initial vaccines for North America.gp120 CM244 is especially preferred for use as the first gp120polypeptide sequence in initial vaccines for Thailand.

In a variation of this embodiment, the vaccine includes two different(i.e., first and second) gp120 polypeptide sequences, or fragmentsthereof, in combination with a breakthrough isolate HIV gp120polypeptide sequence. The latter can be from a vaccinee vaccinated witheither or both of the first and second HIV gp120 polypeptide sequences.

Exemplary vaccines include those containing combinations of gp120 MN,gp120 CM244, gp120 MN-GNE6 (Sequence ID NOs: 43 and 44), and gp120MN-GNE 8 (Sequence ID NO: 46). Combinations of gp120 MN and gp120 CM244or gp120 MN-GNE8 (Sequence ID NO: 46) with a breakthrough isolate HIVgp120polypeptide sequence are especially preferred.

In vaccines containing gp120 MN, the breakthrough isolate HIV gp120polypeptide sequence can be an HIV gp120 polypeptide sequence selectedfrom the group consisting of Sequence ID NOs: 2, 5, 8, 10, 12, 16, 19,23, 25, 28, 31, 33, 36, and 39, and fragments thereof.

The term “subunit vaccine” is used herein, as in the art, to refer to aviral vaccine that does not contain virus, but rather contains one ormore viral proteins or fragments of viral proteins. As used herein, theterm “multivalent”, means that the vaccine contains gp120 from at leasttwo HIV isolates having different amino acid sequences.

The term “breakthrough isolate” or “breakthrough virus” is used herein,as in the art, to refer to a virus isolated from a vaccinee.

The terms “amino acid sequence”, “polypeptide sequence”, and“polypeptide” are used interchangeably herein as in the art, as are theterms “nucleic acid sequence”, “nucleotide sequence”, and“oligonucleotide”.

Polypeptides from Breakthrough Isolates

The gp120 polypeptides of this invention correspond to the amino acidsequences of seven breakthrough isolates which are illustrated below inTable 1. A polypeptide of this invention includes an HIV gp120 aminoacid sequence illustrated in Table 1 (Sequence ID NOs: 1, 4, 7, 9, 11,15, 18, 22, 24, 27, 30, 32, 35, and 38) and fragments thereof. Thepolypeptides of this invention can include fused sequences from two ormore HIV gp120 or gp160 amino acid sequences.

The polypeptide can also be joined to another viral protein, such as aflag epitope amino acid sequence. The term “flag epitope” is usedherein, as in the art, to denote an amino acid sequence that includes anepitope recognized by a monoclonal antibody. Flag epitopes facilitateusing single monoclonal antibody affinity purification of a plurality ofdifferent recombinant proteins, each having the flag epitope recognizedby the monoclonal antibody. Numerous amino acid sequences can functionas flag epitopes. The N-terminal sequences of Herpes Simplex Virus Type1 (HSV-1) glycoprotein D (gD-1) is conveniently used as the flag epitopeand its use is described in detail in the examples. The flag epitope isconveniently fused to the N terminus of the HIV gp120 polypeptidesequence. Alternatively, however, monoclonal antibodies that recognizeneutralizing epitopes in the rgp120 sequences can be used to affinitypurify the amino acid sequences, and a flag epitope can be omitted.

In addition, various signal sequences can be joined to a polypeptide ofthis invention. Although rgp120 is secreted to some extent in HIVcultures, the amount of the envelope glycoprotein released from(secreted by) the host cells varies widely from strain to strain.Various signal sequences can be introduced into the polypeptide byjoining a nucleotide sequence encoding the signal sequence to thenucleotide sequence encoding the rgp120 to facilitate secretion ofrgp120 from the cells. For example, Chiron HIV gp120 polypeptidesinclude a signal sequence from tissue plasminogen activator (TPA) thatprovides good secretion of rgp120. Additional signal sequences are wellknown and include the N-terminal domain of murine leukemia virus surfaceprotein gp70 described by Kayman et al., J. Virol. 68:400–410 (1984).

Table 1 illustrates the nucleotide and deduced amino acid sequences fortwo clones of each the seven breakthrough isolates of this invention.The clones are: C6.1; C6.5; C8.3; C8.6; C15.2; C15.3; C7.2; C7.10;C11.5; C11.7; C10.5; C10.7; C17.1; and C17.3. These sequence are SEQ.ID. NOs: 1–40. The amino acid sequence for MN and the nucleotide anddeduced amino acid sequences for MN-GNE6 and MN-GNE8 are illustrated inthe sequence listing hereinafter. In the listing for MIN-GNE6, a stopcodon appears at amino acid residue position 51. This stop codon can bereplaced with a codon encoding the corresponding amino acid from MN orMN-GNE8 or another isolate.

TABLE 1 CLONE C6.1      GGG GTA CCT GTG TGG AAG GAA GCA ACC ACC ACT CTA36      Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu      1               5                  10 TTT TGT GCA TCA GAT GCT AAAGCA TAT GAC ACA GAG GTG 75 Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp ThrGlu Val           15                  20                  25 CAT AAT GTTTGG GCC ACA CAT GCT TGT GTA CCC ACA GAC 114 His Asn Val Trp Ala Thr HisAla Cys Val Pro Thr Asp                  30                  35 CCA AACCCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA 153 Pro Asn Pro Gln Glu MetVal Leu Glu Asn Val Thr Glu     40                  45                  50 GAT TTT AAC ATG TGG AAAAAT GAC ATG GTA GAA CAG ATG 192 Asp Phe Asn Met Trp Lys Asn Asp Met ValGlu Gln Met              55                  60 CAT GAG GAT ATA ATC AGTTTA TGG GAT CAA AGC CTA AAA 231 His Glu Asp Ile Ile Ser Leu Trp Asp GlnSer Leu Lys  65                  70                  75 CCA TGT GTA AAATTA ACC CCA CTC TGT ATT ACT TTA AAT 270 Pro Cys Val Lys Leu Thr Pro LeuCys Ile Thr Leu Asn          80                  85                  90TGC ACC AAT TGG AAG AAG AAT GAT ACT AAA ACT AAT AGT 309 Cys Thr Asn TrpLys Lys Asn Asp Thr Lys Thr Asn Ser                 95                 100 AGT AGT ACT ACA ACT AAT AAT AGTAGT GCT ACA GCT AAT 348 Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr AlaAsn     105                 110                 115 AGT AGT AGT ACT ACAACT AAT AGT AGT TGG GGA GAG ATA 387 Ser Ser Ser Thr Thr Thr Asn Ser SerTrp Gly Glu Ile             120                 125 AAG GAG GGA GAA ATAAAG AAC TGC TCT TTC AAT ATC ACC 426 Lys Glu Gly Glu Ile Lys Asn Cys SerPhe Asn Ile Thr 130                 135                 140 ACA AGC ATAAGA GAC AAG GTG AAG AAA GAA TAT GCA CTT 465 Thr Ser Ile Arg Asp Lys ValLys Lys Glu Tyr Ala Leu        145                 150                 155 TTT TAT AGC CTT GATGTA GTA CCA ATA GAA AAT GAT AAT 504 Phe Tyr Ser Leu Asp Val Val Pro IleGlu Asn Asp Asn                 160                 165 ACT AGC TAT AGGTTG AGA AGT TGT AAC ACC TCA GTC ATT 543 Thr Ser Tyr Arg Leu Arg Ser CysAsn Thr Ser Val Ile     170                 175                 180 ACACAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC 582 Thr Gln Ala Cys ProLys Val Thr Phe Glu Pro Ile Pro             185                 190 ATACAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG 621 Ile His Tyr Cys ThrPro Ala Gly Phe Ala Ile Leu Lys195                 200                 205 TGT AGA GAT AAA AAG TTC AATGGA ACA GGA CCA TGC AAA 660 Cys Arg Asp Lys Lys Phe Asn Gly Thr Gly ProCys Lys         210                 215                 220 AAT GTT AGCACA GTA CAA TGT GCA CAT GGA ATT AAG CCA 699 Asn Val Ser Thr Val Gln CysAla His Gly Ile Lys Pro                 225                 230 GTA GTGTCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA 738 Val Val Ser Thr Gln LeuLeu Leu Asn Gly Ser Leu Ala    235                 240                 245 GAA GAA GAG GTA ATA ATTAGA TCT GCC AAT TTC TCA AAC 777 Glu Glu Glu Val Ile Ile Arg Ser Ala AsnPhe Ser Asn             250                 255 AAT GCT AAA ATC ATA ATAGTA CAG TTG AGG GAA CCT GTA 816 Asn Ala Lys Ile Ile Ile Val Gln Leu ArgGlu Pro Val 260                 265                 270 GAA ATT AAT TGTACA AGA CCC AGC AAC AAT ACA ATA AAA 855 Glu Ile Asn Cys Thr Arg Pro SerAsn Asn Thr Ile Lys         275                 280                 285GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA 894 Gly Ile His IleGly Pro Gly Arg Ala Phe Tyr Ala Thr                290                 295 GGA GAC ATA CGA GGA GAT ATA AGACAA GCA CAT TGT AAC 933 Gly Asp Ile Arg Gly Asp Ile Ary Gln Ala His CysAsn     300                 305                 310 ATT AGT GGA GCA AAATGG AAT AAC ACT TTA AAG AAG GTA 972 Ile Ser Gly Ala Lys Trp Asn Asn ThrLeu Lys Lys Val             315                 320 GTT AAA AAA TTA AAAGAA CAA TTT CCA AAT AAA ACA ATA 1011 Val Lys Lys Leu Lys Glu Gln Phe ProAsn Lys Thr Ile 325                 330                 335 GTC TTT AACCAT TCC TCA GGA GGG GAC CCA GAA ATT GTA 1050 Val Phe Asn His Ser Ser GlyGly Asp Pro Glu Ile Val        340                 345                 350 ATG CAC AGT TTT AATTGT CAA GGG GAA TTT TTC TAC TGT 1089 Met His Ser Phe Asn Cys Gln Gly GluPhe Phe Tyr Cys                 355                 360 AAT ACA ACA AAGCTG TTT AAT AGT ACT TGG AAT GAT ACT 1128 Asn Thr Thr Lys Leu Phe Asn SerThr Trp Asn Asp Thr     365                 370                 375 ACAGAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA 1167 Thr Glu Ser Asn AsnAsn Asp Ser Thr Ile Thr Leu Pro             380                 385 TGCAGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA ATA 1206 Cys Arg Ile Lys GlnIle Ile Asn Met Trp Gln Glu Ile390                 395                 400 GGA AAA GCA ATG TAT GCC CCTCCC ACC AGA GGA GAA ATT 1245 Gly Lys Ala Met Tyr Ala Pro Pro Thr Arg GlyGlu Ile         405                 410                 415 AAA TGT TCATCA AAT ATT ACA GGA CTA CTG TTA ATA AGA 1284 Lys Cys Ser Ser Asn Ile ThrGly Leu Leu Leu Ile Arg                 420                 425 GAT GGTGGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC 1323 Asp Gly Gly Ile Asn ThrSer Asp Ala Thr Glu Thr Phe    430                 435                 440 AGA CCG GGA GGA GGA GATATG A00 GAC AAT TGG AGA AGT 1362 Arg Pro Gly Gly Gly Asp Met Arg Asp AsnTrp Arg Ser             445                 450 GAA TTA TAT AAA TAT AAAGTA GTG AAA ATT GAG CCA TTA 1401 Glu Leu Tyr Lys Tyr Lys Val Val Lys IleGlu Pro Leu 455                 460                 465 GGA GTA GCA CCCACC AAG GCA AAG AGA AGA GTG GTG CAG 1440 Gly Val Ala Pro Thr Lys Ala LysArg Arg Val Val Gln         470                 475                 480AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT 1479 Arg Glu Lys ArgAla Val Thr Leu Gly Ala Met Phe Leu                485                 490 GGG TTC TTA GGA GCA TAA AGC TTC1503 Gly Phe Leu Gly Ala Xaa Ser Phe     495                 500 501CLONE C6.5     GGG GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA 36    Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu       1               5                  10 TTT TGT GCA TCA GAT GCT AAAGCA TAT GAC ACA GAG GTG 75 Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp ThrGlu Val          15                  20                  25 CAT AAT GTTTGG GCC ACA CAT GCT TGT GTA CCC ACA GAC 114 His Asn Val Trp Ala Thr HisAla Cys Val Pro Thr Asp                  30                 35 CCA AACCCA CAA GAA ATG GTA TTG GAA AAT GTG ACA GAA 153 Pro Asn Pro Gln Glu MetVal Leu Glu Asn Val Thr Glu     40                  45                  50 GAT TTT AAC ATG TGG AAAAAT GAC ATG GTA GAA CAG ATG 192 Asp Phe Asn Met Trp Lys Asn Asp Met ValGlu Gln Met              55                  60 CAT GAG ANT ATA ATC AGTTTA TGG GAT CAA AGC CTA AAA 231 His Glu Xaa Ile Ile Ser Leu Trp Asp GlnSer Leu Lys  65                  70                  75 CCA TGT GTA AAATTA ACC CCA CTC TGT ATT ACT TTA AAT 270 Pro Cys Val Lys Leu Thr Pro LeuCys Ile Thr Leu Asn         80                  85                  90TGC ACC AAT TGG AAG GAG AAT GAT ACT AAA ACT AAT AGT 309 Cys Thr Asn TrpLys Glu Asn Asp Thr Lys Thr Asn Ser                 95                 100 AGT AGT ACT ACA ACT AAT AAT AGTAGT GCT ACA GCT AAT 348 Ser Ser Thr Thr Thr Asn Asn Ser Ser Ala Thr AlaAsn     105                 110                 115 AGT AGT AGT ACT ACAACT AAT AGT AGT TGG GGA GAG ATA 387 Ser Ser Ser Thr Thr Thr Asn Ser SerTrp Gly Glu Ile             120                 125 AAG GAG GGA GAA ATAAAG AAC TGC TCT TTC AAT ATC ACC 426 Lys Glu Gly Glu Ile Lys Asn Cys SerPhe Asn Ile Thr 130                 135                 140 ACA GGC ATAAGA GAC AAG GTG AAG AAA GAA TAT GCA CTT 465 Thr Gly Ile Arg Asp Lys ValLys Lys Glu Tyr Ala Leu        145                 150                 155 TTT TAT AGC CTT GATGTA GTA CCA ATA GAA AAT GAT AAT 504 Phe Tyr Ser Leu Asp Val Val Pro IleGlu Asn Asp Asn                 160                 165 ACT AGC TAT AGGTTG AGA AGT TGT AAC ACC TCA GTC ATT 543 Thr Ser Tyr Arg Leu Arg Ser CysAsn Thr Ser Val Ile     170                 175                 180 ACACAA GCC TGT CCA AAG GTA ACT TTT GAG CCA ATT CCC 582 Thr Gln Ala Cys ProLys Val Thr Phe Glu Pro Ile Pro             185                 190 ATACAT TAT TGT ACC CCG GCT GGT TTT GCG ATT CTG AAG 621 Ile His Tyr Cys ThrPro Ala Gly Phe Ala Ile Leu Lys195                 200                 205 TGT AAA GAT AAA AAG TTC AATGGA ACA GGA CCA TGC AAA 660 Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly ProCys Lys         210                 215                 220 AAT GTT AGCACA GTA CAA TGT ACA CAT GGA ATT AAG CCA 699 Asn Val Ser Thr Val Gln CysThr His Gly Ile Lys Pro                 225                 230 GTA GTGTCA ACT CAA CTG CTG TTA AAT GGC AGC CTA GCA 738 Val Val Ser Thr Gln LeuLeu Leu Asn Gly Ser Leu Ala    235                 240                 245 GAA GAA GAG GTA ATA ATTAGA TCT GCC AAT TTC TCA AAC 777 Glu Glu Glu Val Ile Ile Arg Ser Ala AsnPhe Ser Asn             250                 255 AAT GCT AAA ATC ATA ATAGTA CAG TTG AAG GAA CCT GTA 816 Asn Ala Lys Ile Ile Ile Val Gln Leu LysGlu Pro Val 260                 265                 27 GAA ATT AAT TGTACA AGA CCC AGC AAC AAT ACA ATA AAA 855 Glu Ile Asn Cys Thr Arg Pro SerAsn Asn Thr Ile Lys         275                 280                 285GGT ATA CAC ATA GGA CCA GGG AGA GCA TTT TAT GCA ACA 894 Gly Ile His IleGly Pro Gly Arg Ala Phe Tyr Ala Thr                290                 295 GGA GAC ATA CGA GGA GAT ATA AGACAA GCA CAT TGT AAC 933 Gly Asp Ile Arg Gly Asp Ile Arg Gln Ala His CysAsn     300                 305                 310 ATT AGT GGA GCA AAATGG AAT AAC ACT TTA AAG AAG GTA 972 Ile Ser Gly Ala Lys Trp Asn Asn ThrLeu Lys Lys Val             315                 320 GTT ATA AAA TTA AAAGAA CAA TTT CCA AAT AAA ACA ATA 1011 Val Ile Lys Leu Lys Glu Gln Phe ProAsn Lys Thr Ile 325                 330                 335 GTC TTT AACCAT TCC TCA GGA GGG GAC CCA GAA ATT GTA 1050 Val Phe Asn His Ser Ser GlyGly Asp Pro Glu Ile Val        340                 345                 350 ATG CAC AGT TTT AATTGT CAA GGG GAA TTT TTC TAC TGT 1089 Met His Ser Phe Asn Cys Gln Gly GluPhe Phe Tyr Cys                 355                 360 AAT ACA ACG AAGCTG TTT AAT AGT ACT TGG AAT GAT ACT 1128 Asn Thr Thr Lys Leu Phe Asn SerThr Trp Asn Asp Thr     365                 370                 375 ACAGAG TCA AAT AAC AAT GAT AGT ACT ATT ACA CTC CCA 1167 Thr Glu Ser Asn AsnAsn Asp Ser Thr Ile Thr Leu Pro             380                 385 TGCAGA ATA AAA CAA ATT ATA AAC ATG TGG CAG GAA GTA 1206 Cys Arg Ile Lys GlnIle Ile Asn Met Trp Gln Glu Val390                 395                 400 GGA AAA GCA ATG TAT GCC CCTCCC ATC AGA GGA GAA ATT 1245 Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg GlyGlu Ile         405                 410                 415 AAA TGT TCATCA AAT ATT ACA GGA CTA CTG TTA ACA AGA 1284 Lys Cys Ser Ser Asn Ile ThrGly Leu Leu Leu Thr Arg                 420                 425 GAT GGTGGT ATT AAC ACT AGC GAT GCC ACC GAG ACC TTC 1323 Asp Gly Gly Ile Asn ThrSer Asp Ala Thr Glu Thr Phe    430                 435                 440 AGA CCG GGA GGA GGA GATATG AGG GAC AAT TGG AGA AGT 1362 Arg Pro Gly Gly Gly Asp Met Arg Asp AsnTrp Arg Ser             445                 450 GAA TTA TAT AAA TAT AAAGTA GTG AAA ATT GAG CCA TTA 1401 Glu Leu Tyr Lys Tyr Lys Val Val Lys IleGlu Pro Leu 455                 460                 465 GGA GTA GCA CCCACC AAG GCA AAG AGA AGA GTG GTG CAG 1440 Gly Val Ala Pro Thr Lys Ala LysArg Arg Val Val Gln         470                 475                 480AGA GAA AAA AGA GCA GTA ACA CTA GGA GCT ATG TTC CTT 1479 Arg Glu Lys ArgAla Val Thr Leu Gly Ala Met Phe Leu                485                 490 GGG TTC TTG GGA GCA TAA AGC TTC1503 Gly Phe Leu Gly Ala Xaa Ser Phe     495                 500 501CLONE C8.3 G   GTA CCT GTA TGG AAA GAA GCA ACC ACC ACT CTA TTT 37    Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe      1               5                  10 TGT GCA TCA GAT GCT AAA GCATAT GAT ACA GAG GTA CAT 76 Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr GluVal His          15                  20                  25 AAT GTT TGGGCT ACA CAT GCC TGT GTA CCC ACA GAC CCC 115 Asn Val Trp Ala Thr His AlaCys Val Pro Thr Asp Pro                  30                  35 AAC CCACAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT 154 Asn Pro Gln Glu Val ValLeu Glu Asn Val Thr Glu Asn     40                  45                  50 TTT AAC ATG TGG AAA AATAAC ATG GTA GAA CAG ATG CAT 193 Phe Asn Met Trp Lys Asn Asn Met Val GluGln Met His              55                  60 GAG GAT ATA ATC AGT TTATGG GAT CAA AGT CTA AAG CCA 232 Glu Asp Ile Ile Ser Leu Trp Asp Gln SerLeu Lys Pro  65                  70                 75 TGT GTA AAA TTAACC CCA CTC TGT GTT ACT TTA AAT TGC 271 Cys Val Lys Leu Thr Pro Leu CysVal Thr Leu Asn Cys          80                  85                  90ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT 310 Thr Asn Leu GluAsn Ala Asn Asn Thr Glu Asn Ala Asn                 95                 100 AAT ACC AAT AAT TAT ACC TTG GGGATG GAG AGA GGT GAA 349 Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg GlyGlu     105                 110                 115 ATA AAA AAC TGC TCTTTC AAT ATC ACC ACA AGC TTA AGA 388 Ile Lys Asn Cys Ser Phe Asn Ile ThrThr Ser Leu Arg             120                 125 GAT AAG GTG AAA AAAGAA TAT GCA TTG TTT TAT AAA CTT 427 Asp Lys Val Lys Lys Glu Tyr Ala LeuPhe Tyr Lys Leu 130                 135                 140 GAT GTA GTACAA ATA GAT AAT AGT ACC AAC TAT AGG CTG 466 Asp Val Val Gln Ile Asp AsnSer Thr Asn Tyr Arg Leu        145                 150                 155 ATA AGT TGT AAT ACCTCA OTC ATT ACA CAG GCC TGT CCA 505 Ile Ser Cys Asn Thr Ser Val Ile ThrGln Ala Cys Pro                 160                 165 AAG GTA TCC TTTGAG CTA ATT CCC ATA CAT TAT TGT GCC 544 Lys Val Ser Phe Glu Leu Ile ProIle His Tyr Cys Ala     170                 175                 180 CCGGCT GCT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG 583 Pro Ala Gly Phe AlaIle Leu Lys Cys Lys Asp Lys Lys             185                 190 TTCAAT GGA ACA GGA CCA TGT AAA AAT OTC AGC ACA GTA 622 Phe Asn Gly Thr GlyPro Cys Lys Asn Val Ser Thr Val195                 200                 205 CAA TGT ACA CAT GGA ATT AOACCA GTA GTA TCA ACT CAA 661 Gln Cys Thr His Gly Ile Arg Pro Val Val SerThr Gln         210                 215                 220 CTA CTG TTAAAT GGC AGT CTA GCA GAA GAA GAG ATA GTA 700 Leu Leu Leu Asn Gly Ser LeuAla Glu Glu Glu Ile Val                 225                 230 ATT AGATCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA 739 Ile Arg Ser Glu Asn IleThr Asp Asn Ala Lys Thr Ile    235                 240                 245 ATA GTG CAG CTA AAT GAATCT ATA GTG ATT AAT TGT ACA 778 Ile Val Gln Leu Asn Glu Ser Ile Val IleAsn Cys Thr             250                 255 AGA CCC AAT AAC AAC ACAAGA AAA AGT ATA AAT ATA GGA 817 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleAsn Ile Gly 260                 265                 270 CCA GGG AGA GCATTC TAT ACA ACA GGA GAC ATA ATA GGA 856 Pro Gly Arg Ala Phe Tyr Thr ThrGly Asp Ile Ile Gly         275                 280                 285GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA 895 Asp Ile Arg GlnAla His Cys Asn Leu Ser Lys Thr Gln                290                 295 TGG GAA AAA ACG TTA AGA CAG ATAGCT ATA AAA TTA GAA 934 Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys LeuGlu     300                 305                 310 GAA AAA TTT AAG AATAAA ACA ATA GCC TTT AAT AAA TCC 973 Glu Lys Phe Lys Asn Lys Thr Ile AlaPhe Asn Lys Ser             315                 320 TCA GGA GGG GAC CCAGAA ATT GTA ATG CAC AGT TTT AAT 1012 Ser Gly Gly Asp Pro Glu Ile Val MetHis Ser Phe Asn 325                 330                 335 TGT GGA GGGGAA TTT TTC TAC TGT AAT ACA ACA AAA CTG 1051 Cys Gly Gly Glu Phe Phe TyrCys Asn Thr Thr Lys Leu        340                 345                 350 TTT AAT AGT ACC TGGAAT TTA ACA CAA CCG TTT AGT AAT 1090 Phe Asn Ser Thr Trp Asn Leu Thr GlnPro Phe Ser Asn                 355                 360 ACC GGG AAT CGTACT GAA GAG TTA AAT ATT ACA CTC CCA 1129 Thr Gly Asn Arg Thr Glu Glu LeuAsn Ile Thr Leu Pro     365                 370                 375 TGGAGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA 1168 Cys Arg Ile Lys GlnIle Ile Asn Leu Trp Gln Glu Val             380                 385 GGCAAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT 1207 Gly Lys Ala Met TyrAla Pro Pro Ile Arg Gly Gln Ile390                 395                 400 AGA TGT TCA TCA AAT ATT ACAGGG CTA CTA TTA ACA AGA 1246 Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu LeuThr Arg         405                 410                 415 GAT GGT GGAAGT AAC ACC GGT GAC AAC AGG ACT GAG ACC 1285 Asp Gly Gly Ser Asn Thr GlyAsp Asn Arg Thr Glu Thr                 420                 425 TTT AGACCT GGA GGA GGA GAT ATG AGG GAC AAT TGG AGA 1324 Phe Arg Pro Gly Gly GlyAsp Met Arg Asp Asn Trp Arg    430                 435                 440 AGT GAA TTA TAT AAA TATAAA GTA GTA AGA ATT GAA CCA 1363 Ser Glu Leu Tyr Lys Tyr Lys Val Val ArgIle Glu Pro             445                 450 TTA GGA GTA GCA CCC ACCCAG GCA AAG AGA AGA GTG GTG 1402 Leu Gly Val Ala Pro Thr Gln Ala Lys ArgArg Val Val 455                 460                 465 CAA AGA GAA AAAAGA GCA GTG GGG ATA GGA GCT ATG TTC 1441 Gln Arg Glu Lys Arg Ala Val GlyIle Gly Ala Met Phe         470                 475                 480CTT GGG TTC TTG GGA GAT AA 1461 Leu Gly Phe Leu Gly Asp                 485 486 CLONE C8.6 G   GTA CCT GTG TGG AAA GAA GCA ACCACC ACT CTA TTT 37     Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe      1               5                  10 TGT GCA TCA GAT GCT AAA GCATAT GAT ACA GAG GTA CAT 76 Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr GluVal His          15                  20                  25 AAT GTT TGGGCT ACA CAT GCC TGT GTA CCC ACA GAC CCC 115 Asn Val Trp Ala Thr His AlaCys Val Pro Thr Asp Pro                 30                  35 AAC CCACAA GAA GTA GTA TTG GAA AAT GTA ACA GAA AAT 154 Asn Pro Gln Glu Val ValLeu Glu Asn Val Thr Glu Asn     40                  45                  50 TTT AAC ATG TGG AAA AATAAC ATG GTA GAA CAG ATG CAT 193 Phe Asn Met Trp Lys Asn Asn Met Val GluGln Met His              55                  60 GAG GAT ATA ATC AGT TTATGG GAT CAA AGT CTA AAG CCA 232 Glu Asp Ile Ile Ser Leu Trp Asp Gln SerLeu Lys Pro  65                  70                  75 TGT GTA AAA TTAACC CCA CTC TGT GTT ACT TTA AAT TGC 271 Cys Val Lys Leu Thr Pro Leu CysVal Thr Leu Asn Cys          80                  85                  90ACT AAT TTG GAG AAT GCT AAT AAT ACC GAG AAT GCT AAT 310 Thr Asn Leu GluAsn Ala Asn Asn Thr Glu Asn Ala Asn                 95                 100 AAT ACC AAT AAT TAT ACC TTG GGGATG GAG AGA GGT GAA 349 Asn Thr Asn Asn Tyr Thr Leu Gly Met Glu Arg GlyGlu     105                 110                 115 AGA AAA AAC TGC TCTTTC AAT ATC ACC ACA AGC TTA AGA 388 Arg Lys Asn Cys Ser Phe Asn Ile ThrThr Ser Leu Arg             120                 125 GAT AAG GGG AAA AAAGAA TAT GCA TTG TTT TAT AAA CTT 427 Asp Lys Gly Lys Lys Glu Tyr Ala LeuPhe Tyr Lys Leu 130                 135                 140 GAT GTA GTACAA ATA GAT AAT AGT ACC AAC TAT AGG CTG 466 Asp Val Val Gln Ile Asp AsnSer Thr Asn Tyr Arg Leu        145                 150                 155 ATA AGT TGT AAT ACCTCA GTC ATT ACA CAG GCC TGT CCA 505 Ile Ser Cys Asn Thr Ser Val Ile ThrGln Ala Cys Pro                 160                 165 AAG GTA TCC TTTGAG CCA ATT CCC ATA CAT TAT TGT GCC 544 Lys Val Ser Phe Glu Pro Ile ProIle His Tyr Cys Ala     170                 175                 180 CCGGCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG AAG 583 Pro Ala Gly Phe AlaIle Leu Lys Cys Lys Asp Lys Lys             185                 190 TTCAAT GGA ACA GGA CCA TGT AAA AAT GTC AGG ACA GTA 622 Phe Asn Gly Thr GlyPro Cys Lys Asn Val Arg Thr Val195                 200                 205 CAA TGT ACA CAT GGA ATT AGACCA GTA GTA TCA ACT CAA 661 Gln Cys Thr His Gly Ile Arg Pro Val Val SerThr Gln          210                215                 220 CTA CTG TTAAAT GGC AGT CTA GCA GAA GAA GAG ATA GTA 700 Leu Leu Leu Asn Gly Ser LeuAla Glu Glu Glu Ile Val                 225                 230 ATT AGATCT GAA AAT ATC ACA GAC AAT GCT AAA ACC ATA 739 Ile Arg Ser Glu Asn IleThr Asp Asn Ala Lys Thr Ile    235                 240                 245 ATA GTG GAG CTA AAT GAATCT ATA GTG ATT AAT TGT ACA 778 Ile Val Gln Leu Asn Glu Ser Ile Val IleAsn Cys Thr             250                 255 AGA CCC AAT AAC AAC ACAAGA AAA AGT ATA AAT ATA GGA 817 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleAsn Ile Gly 260                 265                 270 CCA GGG AGA GCATTC TAT ACA ACA GGA GAC ATA ATA GGA 856 Pro Gly Arg Ala Phe Tyr Thr ThrGly Asp Ile Ile Gly         275                 280                 285GAT ATA AGA CAA GCA CAT TGT AAC CTT AGT AAA ACA CAA 895 Asp Ile Arg GlnAla His Cys Asn Leu Ser Lys Thr Gln                290                 295 TGG GAA AAA ACG TTA AGA CAG ATAGCT ATA AAA TTA GAA 934 Trp Glu Lys Thr Leu Arg Gln Ile Ala Ile Lys LeuGlu     300                 305                 310 GAA AAA TTT AAG AATAAA ACA ATA GCC TTT AAT AAA TCC 973 Glu Lys Phe Lys Asn Lys Thr Ile AlaPhe Asn Lys Ser             315                 320 TCA GGA GGG GAC CCAGAA ATT GTA ATG CAC AGT TTT AAT 1012 Ser Gly Gly Asp Pro Glu Ile Val MetHis Ser Phe Asn 325                 330                 335 TGT GGA GGGGGA TTT TTC TAC TGT AGT ACG AGA AAA CTG 1051 Cys Gly Gly Gly Phe Phe TyrCys Ser Thr Arg Lys Leu        340                 345                 350 TTT AAT AGT ACC TGGAAT TTA ACA CAA CCG TTT AGT AAT 1090 Phe Asn Ser Thr Trp Asn Leu Thr GlnPro Phe Ser Asn                 355                 360 ACC GGG GAT CGTACT GAA GAG TTA AAT ATT ACA CTC CCA 1129 Thr Gly Asp Arg Thr Glu Glu LeuAsn Ile Thr Leu Pro     365                 370                 375 TGCAGA ATA AAA CAA ATC ATA AAC TTG TGG CAG GAA GTA 1168 Cys Arg Ile Lys GlnIle Ile Asn Leu Trp Gln Glu Val             380                 385 GGCAAA GCA ATG TAT GCC CCT CCC ATC AGA GGA CAA ATT 1207 Gly Lys Ala Met TyrAla Pro Pro Ile Arg Gly Gln Ile390                 395                 400 AGA TGT TCA TCA AAT ATT ACAGGG CTA CTA TTA AGG AGA 1246 Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu LeuArg Arg         405                 410                 415 GAT GGT GGAAGT AAC ACC AGT GAC AAC CAG ACT GAG ACC 1285 Asp Gly Gly Ser Asn Thr SerAsp Asn Gln Thr Glu Thr                 420                 425 TTT AGACCT GGG GGA GGA GAT ATG AGG GAC AAG TGG AGA 1324 Phe Arg Pro Gly Gly GlyAsp Met Arg Asp Lys Trp Arg    430                 435                 440 AGT GAA TTA TAT AAA TATAAA GTA GTA AGA ATT GAA CCA 1363 Ser Glu Leu Tyr Lys Tyr Lys Val Val ArgIle Glu Pro             445                 450 TTA GGA GTA GCA CCC ACCCAG GCA AAG AGA AGA GTG GTG 1402 Leu Gly Val Ala Pro Thr Gln Ala Lys ArgArg Val Val 455                 460                 465 CAA AGA GAA AAAAGA GCA GTG GGG ATA GGA GCT ATG TTC 1441 Gln Arg Glu Lys Arg Ala Val GlyIle Gly Ala Met Phe         470                 475                 480CTT AGG TTC TTA GGA GAT AAA GCT TCT AGA GTC 1474 Leu Arg Phe Leu Gly AspLys Ala Ser Arg Val                 485                 490 491 CLONEC15.2     CTC GAG GTA CCT GTA TGG AAA GAA GCA ACT ACC ACT 36     Leu GluVal Pro Val Trp Lys Glu Ala Thr Thr Thr      1               5                  10 CTA TTT TGT GCA TCA GAT GCTAAA GCA TAT AAT ACA GAG 75 Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr AsnThr Glu          15                  20                  25 AAA CAT AATGTT TGG GCC ACA CAC GCC TGT GTA CCC ACA 114 Lys His Asn Val Trp Ala ThrHis Ala Cys Val Pro Thr                  30                  35 GAT CCCAAC CCA CAA GAA GTA GTA TTG GGA AAT GTG ACA 153 Asp Pro Asn Pro Gln GluVal Val Leu Gly Asn Val Thr     40                  45                  50 GAA AAT TTT AAC ATG TGGAAA AAT AAC ATG GTA GAA CAA 192 Glu Asn Phe Asn Met Trp Lys Asn Asn MetVal Glu Gln              55                  60 ATG CAT GAA GAT ATA ATCAGT TTA TGG GAT CAA AGT CTA 231 Met His Glu Asp Ile Ile Ser Leu Trp AspGln Ser Leu  65                  70                  75 AAG CCA TGT GTAAAA TTA ACC CCA CTC TGT GTT ACT TTA 270 Lys Pro Cys Val Lys Leu Thr ProLeu Cys Val Thr Leu          80                  85                  90AAT TGC ACT GAT GAT TTA GGG AAT GCT ACT AAT ACC AAT 309 Asn Cys Thr AspAsp Leu Gly Asn Ala Thr Asn Thr Asn                 95                 100 AGT AGT GCC ACT ACC AAT AGT AGTAGT TGG GAA GAA ATG 348 Ser Ser Ala Thr Thr Asn Ser Ser Ser Trp Glu GluMet     105                 110                 115 AAG GGG GAA ATG AAAAGA TGC TCT TTC AAT ATC ACC ACA 387 Lys Gly Glu Met Lys Arg Cys Ser PheAsn Ile Thr Thr             120                 125 AGC ATA AGA GAT AAGATT AAG AAA GAA CAT GCA CTT TTC 426 Ser Ile Arg Asp Lys Ile Lys Lys GluHis Ala Leu Phe 130                 135                 140 TAT AGA CTTGAT GTA GTA CCA ATA GAT AAT GAT AAT ACC 465 Tyr Arg Leu Asp Val Val ProIle Asp Asn Asp Asn Thr         145                  150                155 ACA TAT AGG TTG ATAAAT TGT AAT ACC TCA GTC ATT ACA 504 Thr Tyr Arg Leu Ile Asn Cys Asn ThrSer Val Ile Thr                 160                 165 GAG GCC TGT CGAAAG GTA TCA TTT GAG CCA ATT CCC ATA 543 Gln Ala Cys Pro Lys Val Ser PheGlu Pro Ile Pro Ile     170                 175                 180 CATTTT TGT GCC CGG GCT GGT TTT GCG ATT CTA AAG TGT 582 His Phe Cys Ala ProAla Gly Phe Ala Ile Leu Lys Cys             185                 190 AATAAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT 621 Asn Asn Lys Thr PheGlu Gly Lys Gly Pro Cys Lys Asn195                 200                 205 GTC AGT ACA GTA CAA TGC ACACAT GGA ATT AGG CCA GTA 660 Val Ser Thr Val Gln Cys Thr His Gly Ile ArgPro Val         210                 215                 220 GTG TGA ACTGAA CTG CTG TTA AAT GGC AGT CTA GCA GAA 699 Val Ser Thr Gln Leu Leu LeuAsn Gly Ser Leu Ala Glu                 225                 230 GAA GAGGTA ATA ATT AGA TCT GAG AAT ATC ACA GAG AAT 738 Glu Glu Val Ile Ile ArgSer Asp Asn Ile Thr Asp Asn    235                 240                 245 ACT AAA AGG ATT ATA GTAGAG GTA AAG GAA TGT GTA GTA 777 Thr Lys Thr Ile Ile Val Gln Leu Asn GluSer Val Val             250                 255 ATT AAT TGT AGA AGA CCCAAC AAC AAT ACA AGA AAA AGT 816 Ile Asn Cys Thr Arg Pro Asn Asn Asn ThrArg Lys Ser 260                 265                 270 ATA CAT ATA GGACGA GGG AGT GGA TTT TTT GCA ACA GGA 855 Ile His Ile Gly Pro Gly Ser AlaPhe Phe Ala Thr Gly         275                 280                 285GAA ATA ATA GGA GAT ATA AGA CAA GCA GAG TGT AAC CTT 894 Glu Ile Ile GlyAsp Ile Arg Gln Ala His Cys Asn Leu                290                 295 AGT AGA ACA CAA TGG AAT AAC ACTTTA GGA AAG ATA GTG 933 Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys IleVal     300                 305                 310 ATA AAA TTA AGA GAACAA TTT AGA AAA GAA TTT GGA GAA 972 Ile Lys Leu Arg Glu Gln Phe Arg LysGln Phe Gly Glu             315                 320 AAA ACA ATA GTC TTTAAT CGA TCC TGA GGA GGG GAG CCG 1011 Lys Thr Ile Val Phe Asn Arg Ser SerGly Gly Asp Pro 325                 330                 335 GAA ATT GGAATG GAG AGT TTT AAT TGT GGA GGG GAA TTT 1050 Glu Ile Ala Met His Ser PheAsn Cys Gly Gly Glu Phe        340                 345                 350 TTC TAG TGT AAG AGAACA GGA GTG TTT AAT AGT AGG TGG 1089 Phe Tyr Gys Asn Thr Thr Ala Leu PheAsn Ser Thr Trp                 355                 360 AAT GTT ACT AAAGGG TTG AAT AAC AGT GAA GGA AAT AGG 1128 Asn Val Thr Lys Gly Leu Asn AsnThr Glu Gly Asn Ser     365                 370                 375 ACAGGA GAT GAA AAT ATC ATA CTC GGA TGT AGA ATA AAA 1167 Thr Gly Asp Glu AsnIle Ile Leu Pro Gys Arg Ile Lys             380                 385 CAAATT ATA AAG ATG TGG GAG GAA GTA GGA AAA GGA ATG 1206 Gln Ile Ile Asn MetTrp Gln Glu Val Gly Lys Ala Met390                 395                 400 TAT GGG GGT CCC ATC AGT GGAGAA ATT AGA TGT TGA TGA 1245 Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg GysSer Ser         405                 410                 415 AAG ATT AGAGGG GTG GTA CTA ACA AGA GAT GGT GGT AGT 1284 Asn Ile Thr Gly Leu Leu LeuThr Arg Asp Gly Gly Ser                 420                 425 AAG AACGAG AGC ATC ACC ACC GAG GTC TTC AGA CCT GGA 1323 Lys Asn Glu Ser Ile ThrThr Glu Val Phe Arg Pro Gly    430                 435                 440 GGA GGA GAT ATG AGG GACAAT TGG AGA AGT GAA TTA TAT 1362 Gly Gly Asp Met Arg Asp Asn Trp Arg SerGlu Leu Tyr             445                 450 AAA TAT AAA GTA GTA AAAATT GAA CCA TTA GGA GTA GCG 1401 Lys Tyr Lys Val Val Lys Ile Glu Pro LeuGly Val Ala 455                 460                 465 CCC ACC AAG GCAAAG AGA AGA GTG GTG CAG AGA GAA AAA 1440 Pro Thr Lys Ala Lys Arg Arg ValVal Gln Arg Glu Lys         470                 475                 480AGA GCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC 1479 Arg Ala Val GlyThr Ile Gly Ala Met Phe Leu Gly Phe                485                 490 TTG GGA GCA TAA AGC TTC TAG ACTCGA CCT GCA 1512 Leu Gly Ala Xaa Ser Phe Xaa Ser Arg Pro Ala    495                 500             504 CLONE C15.3     CTC GAG GTACCT GTC TGG AAA GAA GCA ACT ACC ACT 36     Leu Glu Val Pro Val Trp LysGlu Ala Thr Thr Thr       1               5                  10 CTA TTTTGT GCA TCA CAT GCT AAA GCA TAT AAT ACA GAG 75 Leu Phe Cys Ala Ser AspAla Lys Ala Tyr Asn Thr Glu         15                  20                  25 AAA CAT AAT GTT TGGGCC ACA CAC GCC TCT GTA CCC ACA 114 Lys His Asn Val Trp Ala Thr His AlaCys Val Pro Thr                  30                  35 GAT CCC AAC CCACAA GAA CTA GTA TTG GGA AAT GTG ACA 153 Asp Pro Asn Pro Gln Glu Val ValLeu Gly Asn Val Thr      40                  45                  50 GAAAAT TTT AAC ATG TCG AAA AAT AAC ATG GTA GAA CAA 192 Glu Asn Phe Asn MetTrp Lys Asn Asn Met Val Glu Gln              55                  60 ATGCAT GAA GAT ATA ATC AGT TTA TGG CAT CAA AGT CTA 231  Met His Glu Asp IleIle Ser Leu Trp Asp Gln Ser Leu  65                  70                  75 AAG CCA TGT CTA AAA TTA ACCCCA CTC TGT GTT ACT TTA 270 Lys Pro Cys Val Lys Leu Thr Pro Leu Cys ValThr Leu          80                  85                  90 AAT TGC ACTGAT GAT TTA CCC AAT GCT ACT AAT ACC AAT 309 Asn Cys Thr Asp Asp Leu GlyAsn Ala Thr Asn Thr Asn                  95                   100 AGCACT GCC ACT ACC AAT AGT ACT AGT TGC GAA GAA ATG 348 Ser Ser Ala Thr ThrAsn Ser Ser Ser Trp Glu Glu Met    105                 110                     115 AAG GGG GAA ATG AAAAGG TGC TCT TTC AAT ATC ACC ACA 387 Lys Gly Glu Met Lys Arg Cys Ser PheAsn Ile Thr Thr             120                 125 AGC ATA AGA CAT AACATT AAG AAA CAA CAT GCA CTT TTC 426 Ser Ile Arg Asp Lys Ile Lys Lys GluHis Ala Leu Phe 130                 135                 140 TAT AGA CTTGAT CTA GTA CCA ATA CAT AAT CAT AAT ACC 465 Tyr Arg Leu Asp Val Val ProIle Asp Asn Asp Asn Thr        145                 150                 155 ACA TAT AGG TTG ATAAAT TGT AAT ACC TCA GTC ATT ACA 504 Thr Tyr Arg Leu Ile Asn Cys Asn ThrSer Val Ile Thr                 160                 165 CAG CCC TGT CCAAAG GTA TCA TTT GAG CCA ATT CCC ATA 543 Gln Ala Cys Pro Lys Val Ser PheClu Pro Ile Pro Ile     170                 175                 180 CATTTT TCT CCC CCC CCT CCT TTT CCC ATT CTA AAG TCT 582 His Phe Cys Ala ProAla Gly Phe Ala Ile Leu Lys Cys             185                 190 AATAAT AAG ACG TTC GAG GGA AAA GGA CCA TGT AAA AAT 621 Asn Asn Lys Thr PheGlu Gly Lys Gly Pro Cys Lys Asn195                 200                 205 GTC AGT ACA GTA CAA TGC ACACAT GGA ATT AGG CCA GTA 660 Val Ser Thr Val Gln Cys Thr His Gly Ile ArgPro Val         210                 215                 220 GTG TCA ACTCAA CTG CTG TTA AAT GGC AGT CTA GCA GAA 699 Val Ser Thr Gln Leu Leu LeuAsn Gly Ser Leu Ala Glu                 225                 230 GAA GAGGTA ATA ATT AGA TCT GGC AAT ATC ACA GAC AAT 738 Glu Glu Val Ile Ile ArgSer Gly Asn Ile Thr Asp Asn    235                 240                 245 ACT AAA ACC ATT ATA GTACAG CTA AAC GAA TCT GTA GTA 777 Thr Lys Thr Ile Ile Val Gln Leu Asn GluSer Val Val             250                 255 ATT AAT TGT ACA AGA TCCAAC AAC AAT ACA AGA AAA AGT 816 Ile Asn Cys Thr Arg Ser Asn Asn Asn ThrArg Lys Ser 260                 265                 270 ATA CAT ATA GGACCA GGG AGT GCA TTT TTT GCA ACA GGA 855 Ile His Ile Gly Pro Gly Ser AlaPhe Phe Ala Thr Gly         275                 280                 285GAA ATA ATA GGA GAT ATA AGA CAA GCA CAC TGT AAC CTT 894 Glu Ile Ile GlyAsp Ile Arg Gln Ala His Cys Asn Leu                290                 295 AGT AGA ACA CAA TGG AAT AAC ACTTTA GGA AAG ATA GTC 933 Ser Arg Thr Gln Trp Asn Asn Thr Leu Gly Lys IleVal     300                 305                 310 ATA AAA TTA AGA GAACAA TTT AGA AAA CAA TTT GGA GAA 972 Ile Lys Leu Arg Glu Gln Phe Arg LysGln Phe Gly Glu             315                 320 AAA ACA ATA GTC TTTAAT CGA TCC TCA GGA GGG GAC CCG 1011 Lys Thr Ile Val Phe Asn Arg Ser SerGly Gly Asp Pro 325                 330                 335 GAA ATT GCAATG CAC AGT TTT AAT TGT GGA GGG GAA TTT 1050 Glu Ile Ala Met His Ser PheAsn Cys Gly Gly Glu Phe        340                 345                 350 TTC TAC TGT AAC ACAACA GCA CTG TTT AAT AGT ACC TGG 1089 Phe Tyr Cys Asn Thr Thr Ala Leu PheAsn Ser Thr Trp                 355                 360 AAT GTT ACT AAACOG TTG AAT AAC ACT GAA GGA AAT AGC 1128 Asn Val Thr Lys Gly Leu Asn AsnThr Glu Gly Asn Ser     365                 370                 375 ACAGGG GAT GAA AAT ATC ATA CTC CCA TGT AGA ATA AAA 1167 Thr Gly Asp Glu AsnIle Ile Leu Pro Cys Arg Ile Lys             380                 385 CAAATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA ATG 1206 Gln Ile Ile Asn MetTrp Gln Glu Val Gly Lys Ala Met390                 395                 400 TAT GCC CCT CCC ATC AGT GGACAA ATT AGA TGT TCA TCA 1245 Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg CysSer Ser         405                 410                 415 AAT ATT ACACOO CTG CTA CTA ACA AGA GAT GGT GGT AGT 1284 Asn Ile Thr Gly Leu Leu LeuThr Arg Asp Gly Gly Ser                 420                 425 AAG AACGAG ACC ATC ACC ACC GAG GTC TTC AGA CCT GGA 1323 Lys Asn Glu Ser Ile ThrThr Glu Val Phe Arg Pro Gly    430                 435                 440 GGA CGA GAT ATG AGG GACAAT TGG AGA AGT GAA TTA TAT 1362 Gly Gly Asp Met Arg Asp Asn Trp Arg SerGlu Leu Tyr             445             450 AAA TAT AAA GTA GTA AAA ATTGAA CCA TTA GGA GTA GCG 1401 Lys Tyr Lys Val Val Lys Ile Glu Pro Leu GlyVal Ala 455                 460                 465 CCC ACC AAG GCA AAGAGA AGA GTG GTG CAG AGA GAA AAA 1440 Pro Thr Lys Ala Lys Arg Arg Val ValGln Arg Glu Lys         470                 475                 480 AGAGCA GTG GGA ACA ATA GGA GCT ATG TTC CTT GGG TTC 1479 Arg Ala Val Gly ThrIle Gly Ala Met Phe Leu Gly Phe                 485                 490TTA GGA GCA TAA AGC TTC TAG A 1501 Leu Gly Ala Xaa Ser Phe Xaa    495                 500 CLONE C7.2 GG  GAA TTC GGA TCC GGG GTA CCTGTG TGG AAG GAA GCA 38     Glu Phe Gly Ser Gly Val Pro Val Trp Lys GluAla       1               5                  10 ACC ACC ACT CTA TTC TGTGCA TCA GAT GCT AGA GCA TAT 77 Thr Thr Thr Leu Phe Cys Ala Ser Asp AlaArg Ala Tyr          15                  20                  25 GAC ACAGAG GTA CAT AAT GTT TGG GCC ACA CAT GCC TGT 116 Asp Thr Glu Val His AsnVal Trp Ala Thr His Ala Cys                  30                 35 GTACCC ACA GAC CCT AGT CCA CAA GAA GTA GTT TTG GAA 155 Val Pro Thr Asp ProSer Pro Gln Glu Val Val Leu Glu     40                  45                  50 AAT GTG ACA GAA AAT TTTAAC ATG TGG AAA AAT AAC ATG 194 Asn Val Thr Glu Asn Phe Asn Met Trp LysAsn Asn Met              55                  60 GTA GAA CAA ATG CAT GAGGAT ATA ATT AGT TTA TGG GAT 233 Val Glu Gln Met His Glu Asp Ile Ile SerLeu Trp Asp  65                  70                  75 CAA AGC TTA AAGCCA TGT GTA AAA TTA ACC CCA CTC TGT 272 Gln Ser Leu Lys Pro Cys Val LysLeu Thr Pro Leu Cys          80                  85                  90GTT ACT TTA AAT TGC AGT GAT TAT AGG AAT GCT ACT GAT 311 Val Thr Leu AsnCys Ser Asp Tyr Arg Asn Ala Thr Asp                 95                 100 TAT AAG AAT GCT ACT GAT ACC ACTAGT AGT AAC GAG GGA 350 Tyr Lys Asn Ala Thr Asp Thr Thr Ser Ser Asn GluGly     105                 110                 115 AAG ATG GAG AGA GGAGAA ATA AAA AAC TGC TCT TTC AAT 389 Lys Met Glu Arg Gly Glu Ile Lys AsnCys Ser Phe Asn             120                 125 ATT ACC ACA AGC ATAAAA AAT AAG ATG CAG AAA GAA TAT 428 Ile Thr Thr Ser Ile Lys Asn Lys MetGln Lys Glu Tyr 130                 135                 140 GCA CTT TTCTAT AAA CTT GAT ATA GTA CCA ATA GAT AAT 467 Ala Leu Phe Tyr Lys Leu AspIle Val Pro Ile Asp Asn        145                 150                 155 ACA AGC TAT ACA TTGATA AGT TGT AAC ACC TCA GTC ATT 506 Thr Ser Tyr Thr Leu Ile Ser Cys AsnThr Ser Val Ile                 160                      165 ACA CAG GCCTGT CCA AAG GTA TCC TTT GAA CCA ACT CCC 545 Thr Gln Ala Cys Pro Lys ValSer Phe Glu Pro Thr Pro     170                 175                 180ATA CAT TAT TGT GCT CCG GCT GGT TTT GCG ATT CTA AAG 584 Ile His Tyr CysAla Pro Ala Gly Phe Ala Ile Leu Lys             185                 190TGT AAT GAT AAG AAG TTC AGT GGA AAA GGA GAA TGT AAA 623 Cys Asn Asp LysLys Phe Ser Gly Lys Gly Glu Cys Lys195                 200                 205 AAT GTC AGC ACA GTA CAA TGTACA CAT GGA ATT AGG CCA 662 Asn Val Ser Thr Val Gln Cys Thr His Gly IleArg Pro         210                 215                 220 GTA GTA TCAACT CAA CTG CTG TTA AAT GGC AGT CTA GCA 701 Val Val Ser Thr Gln Leu LeuLeu Asn Gly Ser Leu Ala                 225                 230 GAA GAAGAG GTG GTA ATT AGA TCT GAC AAT TTC ATA GAC 740 Glu Glu Glu Val Val IleArg Ser Asp Asn Phe Ile Asp    235                 240                 245 AAT ACT AAA ACC ATA ATAGTA CAG CTG AAA GAA TCT GTA 779 Asn Thr Lys Thr Ile Ile Val Gln Leu LysGlu Ser Val             250                 255 GAA ATT AAT TGT ATA AGACCC AAC AAT AAT ACA AGA AAA 818 Glu Ile Asn Cys Ile Arg Pro Asn Asn AsnThr Arg Lys 260                 265                 270 GGT ATA CAT ATAGGA CCA GGG AGA GCA TGG TAT GCA ACA 857 Gly Ile His Ile Gly Pro Gly ArgAla Trp Tyr Ala Thr         275                 280                 285GGA GAA ATA GTA GGA GAT ATA AGA AAG GCA TAT TGT AAC 896 Gly Glu Ile ValGly Asp Ile Arg Lys Ala Tyr Cys Asn                290                 295 ATT AGT AGA ACA AAA TGG AAT AACACT TTA ATA CAG ATA 935 Ile Ser Arg Thr Lys Trp Asn Asn Thr Leu Ile GlnIle     300                 305                 310 GCT AAC AAA TTA AAAGAA AAA TAT AAT ACA ACA ATA AGC 974 Ala Asn Lys Leu Lys Glu Lys Tyr AsnThr Thr Ile Ser             315                 320 TTT AAT CGA TCC TCAGGA GGG GAC CCA GAA ATT GTA ACG 1013 Phe Asn Arg Ser Ser Gly Gly Asp ProGlu Ile Val Thr 325                 330                 335 CAT AGT TTTAAT TGT GGA GGG GAG TTT TTC TAC TGT GAT 1052 His Ser Phe Asn Cys Gly GlyGlu Phe Phe Tyr Cys Asp        340                 345                 350 TCA ACA CAA CTG TTTAAT AGT ACT TGG AAT TTA AAT GGT 1091 Ser Thr Gln Leu Phe Asn Ser Thr TrpAsn Leu Asn Gly                 355                 360 ACT TGG AAT TTTACT GCA GGG TCA AAT GAA ACT GAA GGC 1130 Thr Trp Asn Phe Thr Ala Gly SerAsn Glu Thr Glu Gly     365                 370                 375 AATATC ACA CTC CCA TGC AGA ATA AAA CAA ATT ATA AAC 1169 Asn Ile Thr Leu ProCys Arg Ile Lys Gln Ile Ile Asn             380                 385 AGGTGG CAG GAA GTA GGG AAA GCA ATG TAT GCC CCT CCC 1208 Arg Trp Gln Glu ValGly Lys Ala Met Tyr Ala Pro Pro390                 395                 400 ATC AGT GGA CAA ATA AAA TGCTCA TCA AAC ATT ACA GGG 1247 Ile Ser Gly Gln Ile Lys Cys Ser Ser Asn IleThr Gly         405                 410                 415 ATG ATA TTAACA AGG GAT GGT GGT AAC GAG AAC AAT AAT 1286 Met Ile Leu Thr Arg Asp GlyGly Asn Glu Asn Asn Asn                 420                 425 GAG AGCAGT ACT ACT GAG ACC TTC AGA CCG GGA GGA GGA 1325 Glu Ser Ser Thr Thr GluThr Phe Arg Pro Gly Gly Gly    430                 435                 440 GAT ATG AGG AAC AAT TGGAGA AGT GAA TTA TAT AAA TAT 1364 Asp Met Arg Asn Asn Trp Arg Ser Glu LeuTyr Lys Tyr             445                 450 AAA GTA GTA AAA ATT GAACCA TTA GGA GTA GCA CCC ACC 1403 Lys Val Val Lys Ile Glu Pro Leu Gly ValAla Pro Thr 455                 460                 465 AAG GCA AAG AGAAGA GTG GTG CAG AGA GAA AAA AGA GCA 1442 Lys Ala Lys Arg Arg Val Val GlnArg Glu Lys Arg Ala         470                 475                 480GTG GGA GCG CTA GGA GCT ATG TTC CTT GGG TTC TTA GGA 1481 Val Gly Ala LeuGly Ala Met Phe Leu Gly Phe Leu Gly                485                 490 GCA TAA AGC TTC TAG ACC GAC TCTAGA GGA TCC 1514 Ala Xaa Ser Phe Xaa Thr Asp Ser Arg Gly Ser    495                 500             504 CLONE C7.10 G   GTA CCT GTGTGG AAG GAA GCA ACC ACC ACT CTA TTC 37     Val Pro Val Trp Lys Glu AlaThr Thr Thr Leu Phe       1               5                 10 TGT GCATCA GAT GCT AGA GCA TAT GAC ACA GAG GTA CAT 76 Cys Ala Ser Asp Ala ArgAla Tyr Asp Thr Glu Val His         15                  20                  25 AAT GTT TGG GCC ACACAT GCC TGT GTA CCC ACA GAC CCT 115 Asn Val Trp Ala Thr His Ala Cys ValPro Thr Asp Pro                  30                  35 AGT CCA CAA GAAGTA TTT TTG GGA AAT GTG ACA GAA AAT 154 Ser Pro Gln Glu Val Phe Leu GlyAsn Val Thr Glu Asn     40                   45                  50 TTTAAT ATG TGG AAA AAT AAC ATG GTA GAA CAA ATG TAT 193 Phe Asn Met Trp LysAsn Asn Met Val Glu Gln Met Tyr              55                  60 GAGGAT ATA ATT AGT TTA TGG GAT CAA AGC TTA AAG CCA 232 Glu Asp Ile Ile SerLeu Trp Asp Gln Ser Leu Lys Pro 65                  70                  75 TGT GTA AAA TTA ACC CCA CTCTGT GTT ACT TTA AAT TGC 271 Cys Val Lys Leu Thr Pro Leu Cys Val Thr LeuAsn Cys          80                  85                  90 AGT GAT TATAGG AAT GCT ACT GAT TAT AAG AAT GCT ACT 310 Ser Asp Tyr Arg Asn Ala ThrAsp Tyr Lys Asn Ala Thr                  95                 100 GAT ACCACT AGT AGT AAC GAG GGA AAG ATG GAG AGA GGA 349 Asp Thr Thr Ser Ser AsnGlu Gly Lys Met Glu Arg Gly    105                 110                 115 GAA ATA AAA AAC TGC TCTTTC AAT ATC ACC ACA AGC ATA 388 Glu Ile Lys Asn Cys Ser Phe Asn Ile ThrThr Ser Ile             120                 125 AAA AAT AAG ATG CAG AAAGAA TAT GCA CTT TTC TAT AAA 427 Lys Asn Lys Met Gln Lys Glu Tyr Ala LeuPhe Tyr Lys 130                 135                 140 CTT AAT ATA GTACCA ATA GAT AAT ACA AGC TAT ACA TTG 466 Leu Asn Ile Val Pro Ile Asp AsnThr Ser Tyr Thr Leu         145                 150                 155ATA AGT TGT AAC ACC TCA GTC ATT ACA CAG GCC TGT CCA 505 Ile Ser Cys AsnThr Ser Val Ile Thr Gln Ala Cys Pro                160                 165 AAG GTA TCC TTT GAA CCA ATT CCCATA CAT TAT TGT GCT 544 Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr CysAla     170                 175                 180 CCG GCT GGT TTT GCGATT CTA AAG TGT AAT GAT AAG AAG 583 Pro Ala Gly Phe Ala Ile Leu Lys CysAsn Asp Lys Lys             185                 190 TTC AGT GGA AAA GGAGAA TGT AAA AAT GTC AGC ACA GTA 622 Phe Ser Gly Lys Gly Glu Cys Lys AsnVal Ser Thr Val 195                 200                 205 CAA TGT ACACAT GGA ATT AGG CCA GTA GTA TCA ACT CAA 661 Gln Cys Thr His Gly Ile ArgPro Val Val Ser Thr Gln        210                 215                 220 CTG CTG TTA AAT GGCAGT CTA GCA GAA GAA GAG GTG GTA 700 Leu Leu Leu Asn Gly Ser Leu Ala GluGlu Glu Val Val                 225                 230 ATT AGA TCT GACAAT TTC ACA GAC AAT ACT AAA ACC ATA 739 Ile Arg Ser Asp Asn Phe Thr AspAsn Thr Lys Thr Ile     235                 240                 245 ATAGTA CAG CTG AAA GAA TCT GTA GAA ATT AAT TGT ATA 778 Ile Val Gln Leu LysGlu Ser Val Glu Ile Asn Cys Ile             250                 255 AGACCC AAC AAT AAT ACA AGA AAA GGT ATA CAT ATA GGA 817 Arg Pro Asn Asn AsnThr Arg Lys Gly Ile His Ile Gly260                 265                 270 CCA GGG AGA GCA TGG TAT GCAACA GGA GAA ATA GTA GGA 856 Pro Gly Arg Ala Trp Tyr Ala Thr Gly Glu IleVal Gly         275                 280                 285 CAT ATA AGACAG GCA TAT TGT AAC ATT AGT AGA ACA AAA 895 Asp Ile Arg Gln Ala Tyr CysAsn Ile Ser Arg Thr Lys                 290                 295 TGG PATAAC ACT TTA ATA CAG ATA GCT AAC AAA TTA AAA 934 Trp Asn Asn Thr Leu IleGln Ile Ala Asn Lys Leu Lys    300                 305                 310 GAA AAA TAT AAT ACA ACAATA AGC TTT PAT CGA TCC TCA 973 Glu Lys Tyr Asn Thr Thr Ile Ser Phe AsnArg Ser Ser             315                 320 GGA GGC GAC CCA CAA ATTGTA ACC CAT AGT TTT PAT TGT 1012 Gly Gly Asp Pro Clu Ile Val Thr His SerPhe Asn Cys 325                 330                 335 GGA GGG GPA TTTTTC TAC TCT PAT TCA ACA CPA CTG TTT 1051 Gly Gly Clu Phe Phe Tyr Cys AsnSer Thr Gln Leu Phe         340                 345                 350PAT AGT ACT TGG PAT TTA PAT COT ACT TGG PAT TTT ACT 1090 Asn Ser Thr TrpAsn Leu Asn Gly Thr Trp Asn Phe Thr                355                 360 GCA GGG TCA PAT GAA ACT CPA CCCPAT ATC ACA CTC CCA 1129 Ala Gly Ser Asn Glu Thr Glu Gly Asn Ile Thr LeuPro     365                 370                 375 TGC AGA ATA AAA CPAATT ATA PAC AGG TGG CAC GPA GTA 1168 Cys Arg Ile Lys Gln Ile Ile Asn ArgTrp Gln Glu Val             380                 385 GGA AAA GCA ATG TATCCC CCT CCC ATC AGT GGA CPA ATA 1207 Gly Lys Ala Met Tyr Ala Pro Pro IleSer Gly Gln Ile 390                 395                 400 AGA TGC TCATCA PAC ATT ACA CCC ATG ATA TTA ACA AGG 1246 Arg Cys Ser Ser Asn Ile ThrGly Met Ile Leu Thr Arg        405                 410                 415 CAT GGT COT PAC GAGPAC PAT PAT GAG AGC ACT ACT ACT 1285 Asp Gly Gly Asn Glu Asn Asn Asn GluSer Ser Thr Thr                 420                 425 GAG ACC TTC ACACCC GGA GGA GGA CAT ATG ACG PAC PAT 1324 Glu Thr Phe Arg Pro Gly Gly GlyAsp Met Arg Asn Asn     430                 435                 440 TCGACA ACT CPA TTA TAT PAA TAT PAA CTA GTA AAA ATT 1363 Trp Arg Ser Glu LeuTyr Lys Tyr Lys Val Val Lys Ile             445                 450 GAGCCA TTA GGA GTA GCA CCC ACC CAC TCT AGA GGA TCC 1402 Glu Pro Leu Gly ValAla Pro Thr Asp Ser Arg Gly Ser455                 460                 465 TCT ACA 1408 Ser Arg     469CLONE C11.5     GAG GTA CCT GTG TCC PAA CPA GCA ACC ACT ACT CTA 36    Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu      1               5                  10 TTT TGT GCA TCA CAT CCT AAAGCA TAT GAC ACA COG GTG 75 Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp ThrGly Val          15                  20                  25 CAT PAT GTTTGG GCC ACA CAT CCC TCT CTA CCC ACA GAC 114 His Asn Val Trp Ala Thr HisAla Cys Val Pro Thr Asp                  30                  35 CCC PACCCA CPA CPA ATA CPA TTG CTA PAT GTG ACA CPA 153 Pro Asn Pro Gln Glu IleGlu Leu Val Asn Val Thr Glu     40                  45                  50 CAT TTT AAC ATG TCC AAAPAT AAA ATC GTA GAC CAG ATG 192 Asp Phe Asn Met Trp Lys Asn Lys Met ValAsp Gln Met              55                  60 CAT GAG GAT ATA ATC AGTTTA TGG GAT GAA AGC CTA AAG 231 His Glu Asp Ile Ile Ser Leu Trp Asp GluSer Leu Lys  65                  70                  75 CCA TGT GTA AAGTTA ACC CCA CTT TGT GTT ACT CTA AAC 270 Pro Cys Val Lys Leu Thr Pro LeuCys Val Thr Leu Asn          80                  85                  90TGC AGT GAT GTG AAC AAT TCC ACA AAT CCT AAT GAT ACT 309 Cys Ser Asp ValAsn Asn Ser Thr Asn Pro Asn Asp Thr                 95                 100 AAT ACT AAT TCC ACT AAT ACT ACTTCC TCT ACT CCT ACG 348 Asn Thr Asn Ser Thr Asn Thr Thr Ser Ser Thr ProThr     105                 110                 115 GCC ACT ACT AGT AGCGAG GAA AAG ATG GAG AAG GGA GAA 387 Ala Thr Thr Ser Ser Glu Glu Lys MetGlu Lys Gly Glu             120                 125 ATA AAA AAC TGC TCTTTC AAT ATC ACC ACA CAC ATG AAA 426 Ile Lys Asn Cys Ser Phe Asn Ile ThrThr His Met Lys 130                 135                 140 GAT AAG GCACAG AAA GAA TAT GCA CTT TTT TAT AAA CTT 465 Asp Lys Ala Gln Lys Glu TyrAla Leu Phe Tyr Lys Leu        145                 150                 155 GAT ATA GTA CCA ATAGAT GAT AAT AAT GCC AGC TAT AGG 504 Asp Ile Val Pro Ile Asp Asp Asn AsnAla Ser Tyr Arg                 160                 165 TTG ATA AGT TGTAAT ACC TCA GAC ATT ACA CAG GCC TGT 543 Leu Ile Ser Cys Asn Thr Ser AspIle Thr Gln Ala Cys     170                 175                 180 CCAAAG GTG ACC TTT GAG CCA ATT CCC ATA CAT TAT TGT 582 Pro Lys Val Thr PheGlu Pro Ile Pro Ile His Tyr Cys             185                 190 GCCCCG GCT GGT TTT GCG ATT CTA AAG TGT AAA GAT AAG 621 Ala Pro Ala Gly PheAla Ile Leu Lys Cys Lys Asp Lys195                 200                 205 AAG TTC AAT GGA ACA GGA CCATGT TCA AAG GTC AGC ACA 660 Lys Phe Asn Gly Thr Gly Pro Cys Ser Lys ValSer Thr         210                 215                 220 GTA CAA TGTACA CAT GGA ATT AGG CCA GTA GTA TCA ACT 699 Val Gln Cys Thr His Gly IleArg Pro Val Val Ser Thr                 225                 230 CAA CTGTTG TTA AAT GGC AGT CTT GCA GAA GAA GAA GTA 738 Gln Leu Leu Leu Asn GlySer Leu Ala Glu Glu Glu Val    235                 240                 245 GTA ATT AGA TCT GTC AATTTC ACA GAC AAT GCT AAA ATC 777 Val Ile Arg Ser Val Asn Phe Thr Asp AsnAla Lys Ile             250                 255 ATA ATA GTA CAG CTG AAAGAA CCT GTA GCA ATT AAT TGT 816 Ile Ile Val Gln Leu Lys Glu Pro Val AlaIle Asn Cys 260                 265                 270 ACA AGA CCC AACAAC AAT ACA AGA AAA GGT ATA CAT CTA 855 Thr Arg Pro Asn Asn Asn Thr ArgLys Gly Ile His Leu         275                 280                 285GGA CCA GGG AGC ACA TTT TAT ACA ACA GGA GAA ATA ATA 894 Gly Pro Gly SerThr Phe Tyr Thr Thr Gly Glu Ile Ile                290                 295 GGA GAC ATA AGA AAA GCA TAT TGCAAG ATT AGT AAA GAA 933 Gly Asp Ile Arg Lys Ala Tyr Cys Lys Ile Ser LysGlu     300                 305                 310 AAA TGG AAT AAC ACTTTA AGA CAG GTA GTT AAA AAA TTA 972 Lys Trp Asn Asn Thr Leu Arg Gln ValVal Lys Lys Leu             315                 320 AGA GAA CAA TTT GGGAAT AAA ACA ATA ATT TTT AAT CGA 1011 Arg Glu Gln Phe Gly Asn Lys Thr IleIle Phe Asn Arg 325                 330                 335 TCC TCA GGAGGG GAC CCA GAA ATT GTA ATG CAC AGT TTT 1050 Ser Ser Gly Gly Asp Pro GluIle Val Met His Ser Phe        340                 345                 350 AAC TGT GGA GGG GAGTTT TTC TAC TGT AAT ACA ACA CAA 1089 Asn Cys Gly Gly Glu Phe Phe Tyr CysAsn Thr Thr Gln                 355                 360 CTG TTT AAT AGTACT TGG AAT AAT ACT GAA GGG ACA AAT 1128 Leu Phe Asn Ser Thr Trp Asn AsnThr Glu Gly Thr Asn     365                 370                 375 AGCACT GAA GGA AAT AGC ACA ATC ACA CTC CCA TGC AGA 1167 Ser Thr Glu Gly AsnSer Thr Ile Thr Leu Pro Cys Arg             380                 385 ATAAAA CAA ATT ATA AAT ATG TGG CAG GAA GTA GGA AAA 1206 Ile Lys Gln Ile IleAsn Met Trp Gln Glu Val Gly Lys390                 395                 400 GCA ACG TAT GCC CCT CCC ATCAGA GGA CGA ATT AGA TGC 1245 Ala Thr Tyr Ala Pro Pro Ile Arg Gly Arg IleArg Cys         405                 410                 415 ATA TCA AATATT ACA GGA CTG CTA TTA ACA AGA GAT GGT 1284 Ile Ser Asn Ile Thr Gly LeuLeu Leu Thr Arg Asp Gly                 420                 425 GGT AGGAAT GTC ACA AAC AAT ACC GAA ACC TTC AGA CCT 1323 Gly Arg Asn Val Thr AsnAsn Thr Glu Thr Phe Arg Pro    430                 435                 440 GGA GGA GGA GAC ATG AGGGAC AAT TGG AGA AGT GAA TTA 1362 Gly Gly Gly Asp Met Arg Asp Asn Trp ArgSer Glu Leu             445                 450 TAT AAA TAT AAA GTA GTAAAA GTT GAA CCA TTA GGA ATA 1401 Tyr Lys Tyr Lys Val Val Lys Val Glu ProLeu Gly Ile 455                 460                 465 GCA CCC ACC AAGGCA AAG AGA AGA GTG GTG CAC AGA GAC 1440 Ala Pro Thr Lys Ala Lys Arg ArgVal Val His Arg Asp         470                 475                 480AAA AGA GCA GCA CTA GGA GCC TTG TTC CTT GGG TTC TTA 1479 Lys Arg Ala AlaLeu Gly Ala Leu Phe Leu Gly Phe Leu                485                 490 GGA GCA TAA AAG CTT CTA GA 1499Gly Ala Xaa Lys Leu Leu      495             499 CLONE C11.7     GAG GTACCT GTA TGG AAA GAA GCA ACC ACT ACT CTA 36     Glu Val Pro Val Trp LysGlu Ala Thr Thr Thr Leu       1               5                  10 TTTTGT GCA TCA GAT GCT AAA GCA TAT GAC ACA GAG GTG 75 Phe Cys Ala Ser AspAla Lys Ala Tyr Asp Thr Glu Val         15                  20                  25 CAT AAT GTT TGG GCCACA CAT GCC TGT GTA CCC ACA GAC 114 His Asn Val Trp Ala Thr His Ala CysVal Pro Thr Asp                  30                  35 CCC AAC CCA CAAGAA ATA GAA TTG GTA AAT GTG ACA GAA 153 Pro Asn Pro Gln Glu Ile Glu LeuVal Asn Val Thr Glu      40                  45                  50 GATTTT AAC ATG TGG AAA AAT AAA ATG GTA GAC CAG ATG 192 Asp Phe Asn Met TrpLys Asn Lys Met Val Asp Gln Met              55                  60 CATGAG GAT ATA ATC AGT TTA TGG GAT GAA AGC CTA AAG 231 His Glu Asp Ile IleSer Leu Trp Asp Glu Ser Leu Lys 65                  70                  75 CCA TGT GTA AAG TTA ACC CCACTT TGT GTT ACT CTA AAC 270 Pro Cys Val Lys Leu Thr Pro Leu Cys Val ThrLeu Asn          80                  85                  90 TGC AGT GATGTG AAC AAT TCC ACA AAT CCT AAT GAT ACT 309 Cys Ser Asp Val Asn Asn SerThr Asn Pro Asn Asp Thr                  95                 100 AAT ACTAAT TCC ACT AAT ACT ACT TCC TCT ACT CCT ACG 348 Asn Thr Asn Ser Thr AsnThr Thr Ser Ser Thr Pro Thr    105                 110                 115 CCC ACT ACT AGT AGC GAGGAA AAG ATG GAG AAG GGA GAA 387 Ala Thr Thr Ser Ser Glu Glu Lys Met GluLys Gly Glu             120                 125 ATA AAA AAC TGC TCT TTCAAT ATC ACC ACA CAC ATG AAA 426 Ile Lys Asn Cys Ser Phe Asn Ile Thr ThrHis Met Lys 130                 135                 140 GAT AAG GTA CAGAAA GAA TAT GCA CTT TTT TAT AAA CTT 465 Asp Lys Val Gln Lys Glu Tyr AlaLeu Phe Tyr Lys Leu         145                 150                 155CAT ATA GTA CCA ATA GAT GAT AAT AAT ACC AGC TAT AGG 504 Asp Ile Val ProIle Asp Asp Asn Asn Thr Ser Tyr Arg                160                 165 TTG ATA AGT TGT AAT ACC TCA GTCATT ACA CAG GCC TGT 543 Leu Ile Ser Cys Asn Thr Ser Val Ile Thr Gln AlaCys     170                 175                 180 CCA ATG GTG ACC TTTGAG CCA ATT CCC ATA CAT TAT TGT 582 Pro Met Val Thr Phe Glu Pro Ile ProIle His Tyr Cys             185                 190 GCC CCG GCT GGT TTTGCG ATT CTA AAG TGT AAA GAT AAG 621 Ala Pro Ala Gly Phe Ala Ile Leu LysCys Lys Asp Lys 195                 200                 205 AAC TTC AATGGA ACA GGA CCA TGT TCA AAG GTC AGC ACA 660 Lys Phe Asn Gly Thr Gly ProCys Ser Lys Val Ser Thr        210                 215                 220 GTA CAA TGT ACA CATGGA ATT AGG CCA GTA GTA TCA ACT 699 Val Gln Cys Thr His Gly Ile Arg ProVal Val Ser Thr                 225                 230 CAA CTG TTG TTAAAT GGC AGT CTT GCA GAA GAA GAA GTA 738 Gln Leu Leu Leu Asn Gly Ser LeuAla Glu Glu Glu Val     235                 240                 245 GTAATT AGA TCT GTC AAT TTC ACA GAC AAT GCT AAA ATC 777 Val Ile Arg Ser ValAsn Phe Thr Asp Asn Ala Lys Ile             250                 255 ATAATA GTA CAG CTG AAA GAA CCT GTA GCA ATT AAT TGT 816 Ile Ile Val Gln LeuLys Glu Pro Val Ala Ile Asn Cys260                 265                 270 ACA AGA CCC AAC AAC AAT ACAAGA AAA GGT ATA CAT CTA 855 Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly IleHis Leu         275                 280                 285 GGA CCA GGGAGC ACA TTT TAT ACA ACA GGA GAA ATA ATA 894 Gly Pro Gly Ser Thr Phe TyrThr Thr Gly Glu Ile Ile                 290                 295 GGA GACATA AGA AAA GCA TAT TGC AAG ATT AGT AAA GAA 933 Gly Asp Ile Arg Lys AlaTyr Cys Lys Ile Ser Lys Glu    300                 305                 310 AAA TGG AAT AAC ACT TTAAGA CAG GTA GTT AAA AAA TTA 972 Lys Trp Asn Asn Thr Leu Arg Gln Val ValLys Lys Leu             315                 320 AGA GAA CAA TTT GGG AATAAA ACA ATA ATT TTT AAT CGA 1011 Arg Glu Gln Phe Gly Asn Lys Thr Ile IlePhe Asn Arg 325                 330                 335 TCC TCA GGA GGGGAC CCA GAA ATT GTA ATG CAC AGT TTT 1050 Ser Ser Gly Gly Asp Pro Glu IleVal Met His Ser Phe         340                 345                 350AAC TGT GGA GGG GAG TTT TTC TAC TGT AAT ACA ACA CAA 1089 Asn Cys Gly GlyGlu Phe Phe Tyr Cys Asn Thr Thr Gln                355                 360 CTG TTT AAT AGT ACT TGG AAT AATACT GAA GGG ACA AAT 1128 Leu Phe Asn Ser Thr Trp Asn Asn Thr Glu Gly ThrAsn     365                 370                 375 AGC ACT GAA GGA AATAGC ACA ATC ACA CTC CCA TGC AGA 1167 Ser Thr Glu Gly Asn Ser Thr Ile ThrLeu Pro Cys Arg             380                 385 ATA AAA CAA ATT ATAAAT ATG TGG CAG GAA GTA GGA AAA 1206 Ile Lys Gln Ile Ile Asn Met Trp GlnGlu Val Gly Lys 390                 395                 400 GCA ACG TATGCC CCT CCC ATC AGA GGA CGA ATT AGA TGC 1245 Ala Thr Tyr Ala Pro Pro IleArg Gly Arg Ile Arg Cys        405                 410                 415 ATA TCA AAT ATT ACAGGA CTG CTA TTA ACA AGA GAT GGT 1284 Ile Ser Asn Ile Thr Gly Leu Leu LeuThr Arg Asp Gly                 420                 425 GGT AGG AAT GTCACA AAC AAT ACC GAN NCC TTC AGA CCT 1323 Gly Arg Asn Val Thr Asn Asn ThrXaa Xaa Phe Arg Pro     430                 435                 440 GGAGGA GGA GAC ATG AGG GAC AAT TGG AGA AGT GAA TTA 1362 Gly Gly Gly Asp MetArg Asp Asn Trp Arg Ser Glu Leu             445                 450 TATAAA TAT AAA GTA GTA AAA GTT GAA CCA TTA GGA ATA 1401 Tyr Lys Tyr Lys ValVal Lys Val Glu Pro Leu Gly Ile455                 460                 465 GCA CCC ACC AAG GCA AAG AGAAGA GTG GTG CAC AGA GAC 1440 Ala Pro Thr Lys Ala Lys Arg Arg Val Val HisArg Asp         470                 475                 480 AAA AGA GCAGCA CTA GGA GCT TTG TTC CTT GGG TTC TTA 1479 Lys Arg Ala Ala Leu Gly AlaLeu Phe Leu Gly Phe Leu                 485                 490 GGA GCATAA AAG CTT CTA GA 1499 Gly Ala Xaa Lys Leu Leu      495             499CLONE C10.5 G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT 37    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe      1                 5                 10 TGT GCA TCA GAT GCT AAA GCATAT GAT AGA GAA GTA CAT 76 Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg GluVal His           15                  20                  25 AAT GTT TGGGCA ACA CAT GCC TGT GTA CCC ACA GAC CCC 115 Asn Val Trp Ala Thr His AlaCys Val Pro Thr Asp Pro                  30                  35 AAC CCACAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT 154 Asn Pro Gln Glu Ile ValLeu Gly Asn Val Thr Glu Asn     40                  45                  50 TTT AAC ATG TGG AAA AATAAC ATG GTA GAA CAA ATG CAT 193 Phe Asn Met Trp Lys Asn Asn Met Val GluGln Met His              55                  60 GAG GAT ATA ATC AAT TTATGG GAT CAA AGC TTA AAG CCA 232 Glu Asp Ile Ile Asn Leu Trp Asp Gln SerLeu Lys Pro  65                  70                 75 TGT GTA AAG TTAACT CCA CTC TGT GTT ACT TTA AAG TGC 271 Cys Val Lys Leu Thr Pro Leu CysVal Thr Leu Lys Cys          80                  85                  90AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT 310 Lys Asp Leu GluArg Aen Thr Thr Tyr Asn Ser Thr Ile                 95                 100 ACC AAT AAT AGT AGT TTG GAG GGACTA AGA GAA CAA ATG 349 Thr Asn Aen Ser Ser Leu Glu Gly Leu Arg Glu GlnMet     105                 110                 115 ACA AAC TGC TCT TTCAAC ATC ACC ACA AGT ATA AGA GAT 388 Thr Asn Cys Ser Phe Asn Ile Thr ThrSer Ile Arg Asp             120                 125 AAG GTG CAG AAA GAATAT GCA CTT TTG TAT AAA CTT GAT 427 Lys Val Gln Lys Glu Tyr Ala Leu LeuTyr Lys Leu Asp 130                 135                 140 GTA GTA CCAATA GAA GAA GAT GAC AAT ACT AGC TAT AGA 466 Val Val Pro Ile Glu Glu AspAsp Asn Thr Ser Tyr Arg        145                 150                 155 TTG ATA AGT TGT AACACC TCA GTC ATT ACA CAG GCT TGT 505 Leu Ile Ser Cys Asn Thr Ser Val IleThr Gln Ala Cys                 160                 165 CCA AAG ACA TCCTTT GAG CCA ATT CCC ATA CAT TAT TGT 544 Pro Lys Thr Ser Phe Glu Pro IlePro Ile His Tyr Cys     170                 175                 180 GCCCCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG 583 Ala Pro Ala Gly PheAla Ile Leu Lys Cys Asn Asp Lys             185                 190 AAGTTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA 622 Lys Phe Asn Gly ThrGly Pro Cys Lys Asn Val Ser Thr195                 200                 205 GTA CAA TGT ACA CAT GGA ATTAGG CCA GTA GTA TCA ACT 661 Val Gln Cys Thr His Gly Ile Arg Pro Val ValSer Thr         210                 215                 220 CAA CTG TTGTTA AAT GGC AGT CTA GCA GAA GAA GAG GTA 700 Gln Leu Leu Leu Asn Gly SerLeu Ala Glu Glu Glu Val                 225                230 GTA ATCAGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC 739 Val Ile Arg Ser Ala AsnPhe Thr Asp Asn Ala Lys Thr    235                 240                 245 ATA ATA GTA CAT CTA AATGAA ACT GTA AAA ATT AAT TGT 778 Ile Ile Val His Leu Asn Glu Thr Val LysIle Asn Cys             250                 255 ACA AGA CTT GGC AAC AATACA AGA AAA AGT ATA AAT ATA 817 Thr Arg Leu Gly Asn Asn Thr Arg Lys SerIle Asn Ile 260                 265                 270 GGA CCA GGG AGAGTA CTC TAT GCA ACA GGA GAA ATA ATA 856 Gly Pro Gly Arg Val Leu Tyr AlaThr Gly Glu Ile Ile         275                 280                 285GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA 895 Gly Asp Ile ArgGln Ala His Cys Asn Ile Ser Arg Ala                290                 295 CAA TGG AAT AAG ACT TTA GAA AAGGTA GTT GAC AAA TTA 934 Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp LysLeu     300                 305                 310 AGA AAA CAA TTT GGGGAT AAT ACA ACA ATA GCT TTT AAT 973 Arg Lys Gln Phe Gly Asp Asn Thr ThrIle Ala Phe Asn             315                 320 CGA TCC TCA GGA GGGGAC CCA GAA ATT GTA ATG CAC ACT 1012 Arg Ser Ser Gly Gly Asp Pro Glu IleVal Met His Thr 325                 330                 335 TTT AAT TGTGGA GGG GAA TTT TTC TAC TGT AAT ACA ACA 1051 Phe Asn Cys Gly Gly Glu PhePhe Tyr Cys Asn Thr Thr        340                 345                 350 CAA CTG TTT AAT AGTACT TGG AAT AAT ACT TGG AAG GAT 1090 Gln Leu Phe Asn Ser Thr Trp Asn AsnThr Trp Lys Asp                 355                 360 CCT AAC AGG AGTGAC AAT ATC ACA CTC CCA TGC AGA ATA 1129 Pro Asn Arg Ser Asp Asn Ile ThrLeu Pro Cys Arg Ile     365                 370                 375 AAACAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA 1168 Lys Gln Ile Ile AsnMet Trp Gln Glu Val Gly Lys Ala             380                 385 ATGTAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA 1207 Met Tyr Ala Pro ProIle Arg Gly Glu Ile Arg Cys Ser390                 395                 400 TCA AAT ATC ACA GGG CTG CTACTA ACA AGA GAT GGT GGT 1246 Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg AspGly Gly         405                 410                 415 AAT GAC GATGGT AAT GAC ACG ACC ACA AAC AGG ACC GAG 1285 Asn Asp Asp Gly Asn Asp ThrThr Thr Asn Arg Thr Glu                 420                 425 ATC TTCAGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG 1324 Ile Phe Arg Pro Gly GlyGly Asp Met Arg Asp Asn Trp    430                 435                 440 AGA AGT GAA TTA TAT AGATAT AAA GTA GTA AAA ATT GAA 1363 Arg Ser Glu Leu Tyr Arg Tyr Lys Val ValLys Ile Glu             445                 450 CCA TTA GGA ATA GCA CCCACC AGG GCA AAG AGA AGA GTG 1402 Pro Leu Gly Ile Ala Pro Thr Arg Ala LysArg Arg Val 455                 460                 465 GTG CAG AGA GAAAAA AGA GCA GTA GGA CTA GGA GCT TTG 1441 Val Gln Arg Glu Lys Arg Ala ValGly Leu Gly Ala Leu         470                 475                 480TTC CTT GGG T TCTTAGGAG CATAAAGCTT CTAGA 1475 Phe Leu Gly         483CLONE C10.7 G   GTA CCT GTG TGG AAA GAA GCA AAC ACA ACT CTA TTT 37    Val Pro Val Trp Lys Glu Ala Asn Thr Thr Leu Phe      1               5                  10 TGT GCA TCA GAT GCT AAA GCATAT GAT AGA GAA GTA CAT 76 Cys Ala Ser Asp Ala Lys Ala Tyr Asp Arg GluVal His          15                  20                  25 AAT GTT TGGGCA ACA CAT GCC TGT GTA CCC ACA GAC CCC 115 Asn Val Trp Ala Thr His AlaCys Val Pro Thr Asp Pro                  30                  35 AAC CCACAA GAA ATA GTA TTG GGA AAT GTG ACA GAA AAT 154 Asn Pro Gln Glu Ile ValLeu Gly Asn Val Thr Glu Asn     40                  45                  50 TTT AAC ATG TGG AAA AATAAC ATG GTA GAA CAA ATG CAT 193 Phe Asn Met Trp Lys Asn Asn Met Val GluGln Met His              55                  60 GAG GAT ATA ATC AAT TTATGG GAT CAA AGC TTA AAG CCA 232 Glu Asp Ile Ile Asn Leu Trp Asp Gln SerLeu Lys Pro  65                  70                  75 TGT GTA AAG TTAACT CCA CTC TGT GTT ACT TTA AAG TGC 271 Cys Val Lys lAu Thr Pro Leu CysVal Thr Leu Lys Cys          80                  85                  90AAG GAT CTG GAG AGG AAT ACT ACC TAT AAT AGC ACT ATT 310 Lys Asp Leu GluArg Asn Thr Thr Tyr Asn Ser Thr Ile                 95                 100 ACC AAT AAT AGT AGT TTG GAG GGACTA AGA GAA CAA ATG 349 Thr Asn Asn Ser Ser Leu Glu Gly Leu Arg Glu GlnMet     105                 110                 115 ACA AAC TGC TCT TTCAAC ATC ACC ACA AGT ATA AGA GAT 388 Thr Asn Cys Ser Phe Asn Ile Thr ThrSer Ile Arg Asp             120                 125 AAG GTG CAG AAA GAATAT GCA CTT TTG TAT AAA CTT GAT 427 Lys Val Gln Lys Glu Tyr Ala Leu LeuTyr Lys Leu Asp 130                 135                 140 GTA GTA CCAATA GAA GAA GAT GAC AAT ACT AGC TAT AGA 466 Val Val Pro Ile Glu 0Th AspAsp Asn Thr Ser Tyr Arg        145                 150                 155 TTG ATA AGT TGT AACACC TCA GTC ATT ACA CAG GCT TGT 505 Leu Ile Ser Cys Asn Thr Ser Val IleThr Gln Ala Cys                 160                 165 CCA AAG ACA TCCTTT GAG CCA ATT CCC ATA CAT TAT TGT 544 Pro Lys Thr Ser Phe Glu Pro IlePro Ile His Tyr Cys     170                 175                 180 GCCCCG GCT GGT TTT GCG ATT CTA AAG TGT AAT GAT AAG 583 Ala Pro Ala Gly PheAla Ile Leu Lys Cys Asn Asp Lys             185                 190 AAGTTC AAT GGA ACA GGA CCA TGT AAA AAT GTC AGC ACA 622 Lys Phe Asn Gly ThrGly Pro Cys Lys Asn Val Ser Thr195                 200                 205 GTA CAA TGT ACA CAT GGA ATTAGG CCA GTA GTA TCA ACT 661 Val Gln Cys Thr His Gly Ile Arg Pro Val ValSer Thr         210                 215                 220 CAA CTG TTGTTA AAT GGC AGT CTA GCA GAA GAA GAG GTA 700 Gln Leu Leu Leu Asn Gly SerLeu Ala Glu Glu Glu Val                 225                 230 GTA ATCAGA TCT GCC AAT TTC ACA GAC AAT GCT AAA ACC 739 Val Ile Arg Ser Ala AsnPhe Thr Asp Asn Ala Lys Thr    235                 240                 245 ATA ATA GTA CAT CTA AATGAA ACT GTA AAA ATT AAT TGT 778 Ile Ile Val His Leu Asn Glu Thr Val LysIle Asn Cys             250                 255 ACA AGA CTT GGC AAC AATACA AGA AAA AGT ATA AAT ATA 817 Thr Arg Leu Gly Asn Asn Thr Arg Lys SerIle Asn Ile 260                 265                 270 GGA CCA GGG AGAGTA CTC TAT GCA ACA GGA GAA ATA ATA 856 Gly Pro Gly Arg Val Leu Tyr AlaThr Gly Glu Ile Ile         275                 280                 285GGA GAC ATA AGA CAA GCA CAT TGT AAC ATT AGT AGA GCA 895 Gly Asp Ile ArgGln Ala His Cys Asn Ile Ser Arg Ala                290                 295 CAA TGG AAT AAG ACT TTA GAA AAGGTA GTT GAC AAG TTA 934 Gln Trp Asn Lys Thr Leu Glu Lys Val Val Asp LysLeu     300                 305                 310 AGA AAA CAA TTT GGGGAT AAT ACA ACA ATA GCT TTT AAT 973 Arg Lys Gln Phe Gly Asp Asn Thr ThrIle Ala Phe Asn             315                 320 CGA TCC TCA GGA GGGGAC CCA GAA ATT GTA ATG CAC ACT 1012 Arg Ser Ser Gly Gly Asp Pro Glu IleVal Met His Thr 325                 330                 335 TTT AAT TGTGGA GGG GAA TTT TTC TAC TGT AAT ACA ACA 1051 Phe Asn Cys Gly Gly Glu PhePhe Tyr Cys Asn Thr Thr        340                 345                 350 CAA CTG TTT AAT AGTACT TGG AAT AAT ACT TGG AAG GAT 1090 Gln Leu Phe Asn Ser Thr Trp Asn AsnThr Trp Lys Asp                 355                 360 CCT AAC AGG AGTGAC AAT ATC ACA CTC CCA TGC AGA ATA 1129 Pro Asn Arg Ser Asp Asn Ile ThrLeu Pro Cys Arg Ile     365                 370                 375 AAACAA ATT ATA AAC ATG TGG CAG GAA GTA GGA AAA GCA 1168 Lys Gln Ile Ile AsnMet Trp Gln Glu Val Gly Lys Ala             380                 385 ATGTAC GCC CCT CCC ATC AGA GGG GAA ATT AGA TGT TCA 1207 Met Tyr Ala Pro ProIle Arg Gly Glu Ile Arg Cys Ser390                 395                 400 TCA AAT ATC ACA GGG CTG CTACTA ACA AGA GAT GGT GGT 1246 Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg AspGly Gly         405                 410                 415 AAT GAC GATGGT AAT GAC ACG ACC ACA AAC AGG ACC GAG 1285 Asn Asp Asp Gly Asn Asp ThrThr Thr Asn Arg Thr Glu                 420                 425 ATC TTCAGA CCT GGA GGA GGA GAT ATG AGG GAC AAT TGG 1324 Ile Phe Arg Pro Gly GlyGly Asp Met Arg Asp Asn Trp    430                 435                 440 AGA AGT GAA TTA TAT AGATAT AAA GTA GTA AAA ATT GAA 1363 Arg Ser Glu Leu Tyr Arg Tyr Lys Val ValLys Ile Glu             445                 450 CCA TTA GGA ATA GCA CCCACC AGG GCA AAG AGA AGA GTG 1402 Pro Leu Gly Ile Ala Pro Thr Arg Ala LysArg Arg Val 455                 460                 465 GTG CAG AGA GAAAAA AGA GCA GTA GGA CTA GGA GCT TTG 1441 Val Gln Arg Glu Lys Arg Ala ValGly Leu Gly Ala Leu         470                 475                 480TTC CTT GGG TTC TTG GGA GCA TAA AGC TTC TAG A 1475 Phe Leu Gly Phe LeuGly Ala Xaa Ser Phe Xaa                 485                 490 491CLONE C17.1     CTC GAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT 36    Leu Glu Val Pro Val Trp Lys Glu Ala Thr Thr Thr      1              5                  10 CTA TTT TGT GCA TCA GAT GCTAAA GCA TAT GAT TCA GAG 75 Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr AspSer Glu          15                  20                  25 GCA CAT AATGTT TGG GCC ACA CAT GCC TGT GTA CCC ACA 114 Ala His Asn Val Trp Ala ThrHis Ala Cys Val Pro Thr                  30                  35 GAC CCCAAC CCA CAA GAA GTA GAA TTG GAA AAT GTG ACA 153 Asp Pro Asn Pro Gln GluVal Glu Leu Glu Asn Val Thr     40                  45                  50 GAA AAT TTT AAC ATG TGGAAA AAT AAC ATG GTA GAA CAG 192 Glu Asn Phe Asn Met Trp Lys Asn Asn MetVal Glu Gln              55                  60 ATG CAT GGO GAT ATA ATTAGT TTA TGG GAT CAA AGC CTA 231 Met His Gly Asp Ile Ile Ser Leu Trp AspGln Ser Leu  65                  70                  75 AAG CCA TGT GTAAAA TTA ACC CCA CTC TGT GTT ACG TTA 270 Lys Pro Cys Val Lys Leu Thr ProLeu Cys Val Thr Leu          80                  85                  90AAT TGC ACT GAC CCA AAT GTT ACT AAT AGC GAG AGA ACG 309 Asn Cys Thr AspPro Asn Val Thr Asn Ser Glu Arg Thr                 95                 100 ATA GAG GGG GGA GAA ATA AAA AATTGC TCT TTC AAT ATC 348 Ile Glu Gly Gly Glu Ile Lys Asn Cys Ser Phe AsnIle     105                 110                 115 ACC ACA AAC ATA AGAGAT AGG TTT CAG AAA GAA TAT GCA 387 Thr Thr Asn Ile Arg Asp Arg Phe GlnLys Glu Tyr Ala             120                 125 CTT TTT TAT AAA CTTGAT GTA ATA CCA TTA GGT AAT GAT 426 Leu Phe Tyr Lys Leu Asp Val Ile ProLeu Gly Asn Asp 130                 135                 140 AAT ACT AGCTAT AGG TTG ATA AGT TGT AAC ACC TCA GTC 465 Asn Thr Ser Tyr Arg Leu IleSer Cys Asn Thr Ser Val        145                 150                 155 ATT ACA CAG GCC TGTCCA AAG GTA TCC TTT GAG CCA ATT 504 Ile Thr Gln Ala Cys Pro Lys Val SerPhe Glu Pro Ile                 160                 165 CCC ATA CAT TATTGT GCC CCG GCT GGT TTT GCG ATT CTA 543 Pro Ile His Tyr Cys Ala Pro AlaGly Phe Ala Ile Leu     170                 175                 180 AAGTGT AAA GAT AAG AAG TTC AAT GGA ACA GGA CCA TGT 582 Lys Cys Lys Asp LysLys Phe Asn Gly Thr Gly Pro Cys             185                 190 ACAAAT GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AAG 621 Thr Asn Val Ser ThrVal Gln Cys Thr His Gly Ile Lys195                 200                 205 CCA GTA GTA TCA ACT CAA CTGTTG TTA AAT GGC AGT CTA 660 Pro Val Val Ser Thr Gln Leu Leu Leu Asn GlySer Leu         210                 215                 220 GCA GAA GAAGAC ATA GTA ATT AGA TCC GCC AAT CTC ACA 699 Ala Glu Glu Asp Ile Val IleArg Ser Ala Asn Leu Thr                 225                 230 GAC AATGCT AAA AAC ATA ATA GTA CAG CTG AAT GAA TCT 738 Asp Asn Ala Lys Asn IleIle Val Gln Leu Asn Glu Ser    235                 240                 245 GTA ACA ATG AAT TGT ACAAGA CCC AAC AAC AAT ACA ATG 777 Val Thr Met Asn Cys Thr Arg Pro Asn AsnAsn Thr Met             250                 255 AAA AGT ATA CAT ATA GGACCA GGC AGA GCA TTT TAT GCA 816 Lys Ser Ile His Ile Gly Pro Gly Arg AlaPhe Tyr Ala 260                 265                 270 ACA GGA AAC ATAATA GGA GAT ATA AGA CAA GCA CAT TGT 855 Thr Gly Asn Ile Ile Gly Asp IleArg Gln Ala His Cys         275                 280                 285AAC ATT AGT GGA ACA AAA TGG AAT GAC ACT TTG AAA AAG 894 Asn Ile Ser GlyThr Lys Trp Asn Asp Thr Leu Lys Lys                290                 295 ATA GCT ATA AAA TTA AGA GAA CAATTT AAT AAG ACA ATA 933 Ile Ala Ile Lys Leu Arg Glu Gln Phe Asn Lys ThrIle     300                 305                 310 GTC TTT AAT CAA TCCTCA GGA GGG GAC CCA GAA ATT GCA 972 Val Phe Asn Gln Ser Ser Gly Gly AspPro Glu Ile Ala             315                 320 ACG CTC AGT TTT AATTGT GGA GGG GAA TTT TTC TAC TGT 1011 Thr Leu Ser Phe Asn Cys Gly Gly GluPhe Phe Tyr Cys 325                 330                 335 AAT TCA ACACAA CTG TTT AAT AGT ACT TGG AAT AGT ACT 1050 Asn Ser Thr Gln Leu Phe AsnSer Thr Trp Asn Ser Thr        340                 345                 350 GGG TCA AAT AAC ACTAAA GGA AAT GAC ACA ATC ACA CTC 1089 Gly Ser Asn Asn Thr Lys Gly Asn AspThr Ile Thr Leu                 355                 360 CCA TGC AGA ATAAGA CAA ATT ATA AAC ATG TGG CAG AAA 1128 Pro Cys Arg Ile Arg Gln Ile IleAsn Met Trp Gln Lys     365                 370                 375 ATAGGA AAA GCA ATG TAT GCC CCT CCC ATC AAA GGG CAA 1167 Ile Gly Lys Ala MetTyr Ala Pro Pro Ile Lys Gly Gln             380                 385 ATTAGA TGT TCA TCA AAT ATT ACA GGG CTA ATA TTA ACA 1206 Ile Arg Cys Ser SerAsn Ile Thr Gly Leu Ile Leu Thr390                 395                 400 AGA GAT GGT GGT AAC AAC AACATG AGC AAG ACC ACC GAG 1245 Arg Asp Gly Gly Asn Asn Asn Met Ser Lys ThrThr Glu         405                 410                 415 ACC TTC AGACCT GGA GGA GGA GAT ATG AOG GAC AAT TGG 1284 Thr Phe Arg Pro Gly Gly GlyAsp Met Arg Asp Asn Trp                 420                 425 AGA AGTGAA TTA TAT AAA TAT AAA GTA GTA AAA ATT GAA 1323 Arg Ser Glu Leu Tyr LysTyr Lys Val Val Lys Ile Glu    430                 435                 440 CCA TTA GGA GTA GCA CCCACC AGG GCA AAG AGA AGA GTG 1362 Pro Leu Gly Val Ala Pro Thr Arg Ala LysArg Arg Val             445                 450 GTG CAG AGA GAA AAA AGAGCA GTG GGA ATA GGA GCT GTG 1401 Val Gln Arg Glu Lys Arg Ala Val Gly IleGly Ala Val 455                 460                 465 TTC CTT GGG TTCTTG GGA GCA TAA AGC TTC TAG A 1435 Phe Leu Gly Phe Leu Gly Ala Xaa SerPhe Xaa         470                 475         478 CLONE C17.3     CTCGAG GTA CCT GTG TGG AAA GAA GCA ACC ACC ACT 36     Leu Glu Val Pro ValTrp Lys Glu Ala Thr Thr Thr       1               5                  10CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAT TCA GAG 75 Leu Phe Cys AlaSer Asp Ala Lys Ala Tyr Asp Ser Glu          15                 20                  25 GCA CAT AAT GTT TGGGCC ACA CAT GCC TGT GTA CCC ACA 114 Ala His Asn Val Trp Ala Thr His AlaCys Val Pro Thr                  30                  35 GAC CCC AAC CCACAA GAA GTA GAA TTG GAA AAT GTG ACA 153 Asp Pro Asn Pro Gln Glu Val GluLeu Glu Asn Val Thr      40                  45                  50 GAAAAT TTT AAC ATG TGG AAA AAT AAC ATG GTA GAA CAG 192 Glu Asn Phe Asn MetTrp Lys Asn Asn Met Val Glu Gln              55                  60 ATGCAT GGG GAT ATA ATT AGT TTA TGG GAT CAA AGC CTA 231 Met His Gly Asp IleIle Ser Leu Trp Asp Gln Ser Leu 65                  70                  75 AAG CCA TGT GTA AAA TTA ACCCCA CTC TGT GTT ACG TTA 270 Lys Pro Cys Val Lys Leu Thr Pro Leu Cys ValThr Leu          80                  85                  90 AAT TGC ACTGAC CCA AAT GTT ACT AAT AGC GAG AGA ACG 309 Asn Cys Thr Asp Pro Asn ValThr Asn Ser Glu Arg Thr                  95                 100 ATA GAGGGG GGA GAA ATA AAA AAT TGC TCT TTC AAT ATC 348 Ile Glu Gly Gly Glu IleLys Asn Cys Ser Phe Asn Ile    105                 110                 115 ACC ACA AAC ATA AGA GATAGG TTT CAG AAA GAA TAT GCA 387 Thr Thr Asn Ile Arg Asp Arg Phe Gln LysGlu Tyr Ala             120                 125 CTT TTT TAT AAA CTT GATGTA ATA CCA TTA GGT AAT GAT 426 Leu Phe Tyr Lys Leu Asp Val Ile Pro LeuGly Asn Asp 130                 135                 140 AAT ACT AGC TATAGG TTG ATA AGT TGT AAC ACC TCA GTC 465 Asn Thr Ser Tyr Arg Leu Ile SerCys Asn Thr Ser Val         145                 150                 155ATT ACA CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT 504 Ile Thr Gln AlaCys Pro Lys Val Ser Phe Glu Pro Ile                160                 165 CCC ATA CAT TAT TGT GCC CCG GCTGGT TTT GCG ATT CTA 543 Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala IleLeu     170                 175                 180 AAG TGT AAA GAT AAGAAG TTC AAT GGA ACA GGA CCA TGT 582 Lys Cys Lys Asp Lys Lys Phe Asn GlyThr Gly Pro Cys             185                 190 ACA AAT GTC AGC ACAGTA CAA TGT ACA CAT GGA ATT AAG 621 Thr Asn Val Ser Thr Val Gln Cys ThrHis Gly Ile Lys 195                 200                 205 CCA GTA GTATCA ACT CAA CTG TTG TTA AAT GGC AGT CTA 660 Pro Val Val Ser Thr Gln LeuLeu Leu Asn Gly Ser Leu        210                 215                 220 GCA GAA GAA GAC ATAGTA ATT AGA TCC GCC AAT CTC ACA 699 Ala Glu Glu Asp Ile Val Ile Arg SerAla Asn Leu Thr                 225                 230 GAC AAT GCT AAAAAC ATA ATA GTA CAG CTG AAT GAA TCT 738 Asp Asn Ala Lys Asn Ile Ile ValGln Leu Asn Glu Ser     235                 240                 245 GTAACA ATG AAT TGT ACA AGA CCC AAC AAC AAT ACA ATG 777 Val Thr Met Asn CysThr Arg Pro Asn Asn Asn Thr Met             250                 255 AAAAGT ATA CAT ATA GGA CCA GGC AGA GCA TTT TAT GCA 816 Lys Ser Ile His IleGly Pro Gly Arg Ala Phe Tyr Ala260                 265                 270 ACA GGA AAC ATA ATA GGA GATATA AGA CAA GCA CAT TGT 855 Thr Gly Asn Ile Ile Gly Asp Ile Arg Gln AlaHis Cys         275                 280                 285 AAC ATT AGTGGA ACA AAA TGG AAT GAC ACT TTG AAA AAG 894 Asn Ile Ser Gly Thr Lys TrpAen Asp Thr Leu Lys Lys                 290                 295 ATA GCTATA AAA TTA AGA GAA CAA TTT AAT AAG ACA ATA 933 Ile Ala Ile Lys Leu ArgGlu Gln Phe Asn Lys Thr Ile    300                 305                 310 GTC TTT AAT CAA TCC TCAGGA GGG GAC CCA GAA ATT GCA 972 Val Phe Asn Gln Ser Ser Gly Gly Asp ProGlu Ile Ala             315                 320 ACG CTC AGT TTT AAT TGTGGA GGG GAA TTT TTC TAC TGT 1011 Thr Leu Ser Phe Asn Cys Gly Gly Glu PhePhe Tyr Cys 325                 330                 335 AAT TCA ACA CAACTG TTT AAT AGT ACT TGG AAT AGT ACT 1050 Asn Ser Thr Gln Leu Phe Asn SerThr Trp Asn Ser Thr         340                 345                 350GGG TCA AAT AAC ACT AAA GGA AAT GAC ACA ATC ACA CTC 1089 Gly Ser Asn AsnThr Lys Gly Asn Asp Thr Ile Thr Leu                355                 360 CCA TGC AGA ATA AGA CAA ATT ATAAAC ATG TGG CAG AAA 1128 Pro Cys Arg Ile Arg Gln Ile Ile Asn Met Trp GlnLys     365                 370                 375 ATA GGA AAA GCA ATGTAT GCC CCT CCC ATC AAA GGG CAA 1167 Ile Gly Lys Ala Met Tyr Ala Pro ProIle Lys Gly Gln             380                 385 ATT AGA TGT TCA TCAAAT ATT ACA GGG CTA ATA TTA ACA 1206 Ile Arg Cys Ser Ser Asn Ile Thr GlyLeu Ile Leu Thr 390                 395                 400 AGA GAT GGTGGT AAC AAC AAC ATG AGC AAG ACC ACC GAG 1245 Arg Asp Gly Gly Asn Asn AsnMet Ser Lys Thr Thr Glu        405                 410                 415 ACC TTC AGA CCT GGAGGA GGA GAT ATG AGG GAC AAT TGG 1284 Thr Phe Arg Pro Gly Gly Gly Asp MetArg Asp Asn Trp                 420                 425 AGA AGT GAA TTATAT AAA TAT AAA GTA GTA AAA ATT GAA 1323 Arg Ser Glu Leu Tyr Lys Tyr LysVal Val Lys Ile Glu     430                 435                 440 CCATTA GGA GTA GCA CCC ACC AGG GCA AAG AGA AGA GTG 1362 Pro Leu Gly Val AlaPro Thr Arg Ala Lys Arg Arg Val             445                 450 GTGCAG AGA GAA AAA AGA GCA GTG GGA ATA GGA GCT GTG 1401 Val Gln Arg Glu LysArg Ala Val Gly Ile Gly Ala Val455                 460                 465 TTC CTT GGG TTC TTG GGA GCATAA AGC TTC TAG A 1435 Phe Leu Gly Phe Leu Gly Ala Xaa Ser Phe Xaa        470                 475         478

In addition to the listing in Table 1, FIG. 3 shows the alignment of theamino acid sequences of the clones of each of the seven isolates.Corresponding residues from various clones are in boxes. In the figure,the amino acid sequences are aligned against MN-rgp120 (SEQ. ID. NO: 41.

In one embodiment, a gp120 polypeptide of this invention has the sameamino acid sequence as the sequence of one of the breakthrough isolates.In another embodiment, the amino acid sequence is truncated, asdescribed in detail hereinafter. In another embodiment, a gp120polypeptide sequence of this invention contains a substitution,insertion, or deletion (alteration) of one or more amino acids in thesequence of a breakthrough isolate. Usually, with the exception of aminoacids that are not present in a truncated amino acid sequence andeliminate an epitope, a gp120 polypeptide of this invention will includealterations in the amino acid sequence of a breakthrough isolate that donot alter the polypeptide's ability to induce the same neutralizingantibodies as the amino acid sequence of the isolate.

In general, substitutions in the amino acid sequence of a gp120polypeptide of this invention are conservative substitutions,particularly for amino acid residues in the V2, V3, and C4 domains ofgp120, which domains contain neutralizing epitopes. However,non-conservative substitutions, particularly in domains that do notcontain neutralizing epitopes are contemplated.

Conservative substitutions replace an amino acid with an amino acid ofsimilar size and character. For example, a hydrophobic residue orhydrophilic residue is replaced with another hydrophobic residue orhydrophilic residue, respectively. Amino acids can be divided into thefollowing groups: positively charged residues (K, R and H); negativelycharged residues (D and E); amides (N and Q); aromatics (F, Y, and W);hydrophobics (P, G, A, V, L, I, and M); and uncharged residues (S andT). Usually, residues within a group are replaced with another member ofthe group.

In one embodiment, critical amino acid residues in the V2, V3, and C4domains of gp120 are identical to the corresponding residues in abreakthrough-isolate sequence. Critical amino acid residues in the V2,V3, and C4 domains of gp120 are described in the experimental section.In another embodiment, all amino acid residues in the V2, V3, and C4domains of gp120 are identical to corresponding residues in abreakthrough isolate sequence.

Oligonucleotide Encoding gp120 from Breakthrough Isolates

The present invention also provides novel oligonucleotides encodinggp120 from the breakthrough isolates which can be used to express gp120.An oligonucleotide of this invention encodes a polypeptide of thisinvention. The oligonucleotide can be DNA or RNA, usually DNA. Althoughnumerous nucleotide sequences can encode the same amino acid sequencedue to the degeneracy of the genetic code, conveniently, theoligonucleotides of this invention include a nucleotide sequence of abreakthrough isolate as illustrated in Table 1 (Sequence ID NOs: 2, 5,8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36). Usually, an oligonucleotideof this invention is less than about 5 kilobases (kb), preferably lessthan about 3 kb.

To express the encoded amino acid sequence, the oligonucleotide can beinserted into a transcription unit. The transcription unit can beinserted into a plasmid for production of cell lines, inserted into avirus (e.g.; vaccinia) or can be used directly as a DNA vaccine.Suitable transcription units for production of vaccine proteins are wellknown. A preferred expression vector, designated psvI6B5, is illustratedin Sequence ID NO: 45. The vector includes an HSV-1 gDl signal sequencejoined to a linker sequence. The gp120 nucleotide sequence to beexpressed starts with the Kpn I site of the gene. Since all gp120 orgp160 sequences contain this site, any gp120 nucleotide sequence can beanalogously inserted into the vector and expressed. The vector ends witha poly A tail from SV40.

In addition to being useful to express a polypeptide sequence of thisinvention, the oligonucleotides of this invention can also be used indiagnostics to detect HIV isolates. For example, the oligonucleotide ora portion thereof encoding a neutralizing epitope can be used inbranched chain DNA diagnostics or as a probe in in situ hybridizationstudies.

Vaccine Preparation

A gp120 polypeptide of this invention from a selected breakthroughisolate(s) in a suitable carrier is used to make a subunit vaccine. Thepolypeptide can be used alone, but is generally administered in amultivalent subunit vaccine that includes gp120 MN. In addition to oneor more gp120 polypeptides of this invention, the vaccine generallyincludes the MN polypeptide (hereinafter, MN-rgp120). The vaccineusually includes about 3 to about 5 different gp120 polypeptides, but 30or more different gp120 polypeptides can be used.

Preparation of gp120 polypeptides for use in a vaccine is well known andis described hereinafter. With the exception of the use of the selectedHIV isolate, the gp120 subunit vaccine prepared in the method does notdiffer from gp120 subunit vaccines of the prior art.

As with prior art gp120 subunit vaccines, gp120 at the desired degree ofpurity and at a sufficient concentration to induce antibody formation ismixed with a physiologically acceptable carrier. A physiologicallyacceptable carrier is nontoxic to a recipient at the dosage andconcentration employed in the vaccine. Generally, the vaccine isformulated for injection, usually intramuscular or subcutaneousinjection. Suitable carriers for injection include sterile water, butpreferably are physiologic salt solutions, such as normal saline orbuffered salt solutions such as phosphate-buffered saline or ringer'slactate. The vaccine generally contains an adjuvant. Useful adjuvantsinclude QS21 (Quillaja saponaria, commercially available from CambridgeBiotech, Worcester, Mass.), which stimulates cytotoxic T-cells, and alum(aluminum hydroxide adjuvant). Formulations with different adjuvantswhich enhance cellular or local immunity can also be used. Inparticular, immunopotentiators such as cytokines can be included in thevaccine. Examples of suitable immunopotentiating cytokines includeinterleukins, such as interleukin-2 (IL-2) and interleukin-12 (IL-12),and tumor necrosis factor-alpha (TNF-α).

Additional excipients that can be present in the vaccine include lowmolecular weight polypeptides (less than about 10 residues), proteins,amino acids, carbohydrates including glucose or dextrans, chelatingagents such as EDTA, and other excipients that stabilize the protein orinhibit growth of microorganisms.

The vaccine can also contain other HIV proteins. In particular, gp41 orthe extracellular portion of gp41 or HIV-1 core proteins such as P24,P17, and P55 can be present in the vaccine. Although the amino acidsequence of gp41 is more conserved than that of gp120, gp41 containsneutralizing epitopes. Preferably, any gp41 present in the vaccine isfrom an HIV isolate present in the vaccine. gp160 from an isolate usedin the vaccine can replace gp120 in the vaccine or be used together withgp120 from the isolate. Alternatively, gp160 from a different isolatethan those in the vaccine can additionally be present in the vaccine.

Vaccines according to the invention can also contain one or more solublegp120 polypeptide sequences, or fragments thereof, in combination withan engineered virus specifically designed to express proteins thatinduce a cytotoxic T-cell response. Suitable engineered viruses arederived from, for example, Canary Pox virus, vaccinia viruses,attenuated human herpes viruses (such as, e.g., herpes simplex viruses),and Varicella Zoster. Exemplary engineered viruses are modified toexpress any HIV protein capable of inducing a cytotoxic T-cell response,such as those described above. Typically, immunization with thegp120/engineered virus vaccine is followed by administration of one ormore doses of the gp120 polypeptide sequence(s) to boost the immuneresponse. If desired, viruses can be engineered to express one or moregp120 polypeptide sequences of the invention, or fragments thereof, andused in vaccines with or without soluble gp120 polypeptide sequences.

Vaccine formulations generally include a total of about 300 to 600 .mu.gof gp120, conveniently in about 1.0 ml of carrier. Preferredformulations include use of twice the weight of a gp120 polypeptide intwice as 600 .mu.g alum. However, formulations having smaller amounts(e.g.; 50 .mu.g per dose) are also used, generally with alum or otheradjuvants. The amount of gp120 for any isolate present in the vaccinewill vary depending on the immunogenicity of the gp120. For example,gp120 from some strains of HIV may be less immunogenic than gp120 fromthe MN strain (Sequence ID NO: 41). If two strains having differentimmunogenicity are used in combination, empirical titration of theamount of each virus would be performed to determine the percent of thegp120 of each strain in the vaccine. For isolates having similarimmunogenicity, approximately equal amounts of each isolate's gp120would be present in the vaccine. For example, in a preferred embodiment,the vaccine includes gp120 from the MN and a strain of this invention atconcentrations of about 300 .mu.g per strain in about 1.0 ml of carrier.When the vaccine includes gp120 from about 30 isolates, about 10 toabout 50 .mu.g can be used. Methods of determining the relative amountof an immunogenic protein in multivalent vaccines are well known andhave been used, for example, to determine relative proportions ofvarious isolates in multivalent polio vaccines.

The vaccines of this invention are administered in the same manner asprior art HIV gp120 subunit vaccines. In particular, the vaccines aregenerally administered at 0, 1, and at 6, 8 or 12 months, depending onthe protocol. A preferred protocol includes administration at 0, 1, 6,and 12 months. Following the immunization procedure, annual or bi-annualboosts can be administered. However, during the immunization process andthereafter, neutralizing antibody levels can be assayed and the protocoladjusted accordingly.

The vaccine is administered to uninfected individuals. In addition, thevaccine can be administered to seropositive individuals to augmentimmune response to the virus, as with prior art HIV vaccines. It is alsocontemplated that DNA encoding the strains of gp120 for the vaccine canbe administered in a suitable vehicle for expression in the host. Inthis way, gp120 can be produced in the infected host, eliminating theneed for repeated immunizations. Preparation of gp120 expressionvehicles is described hereinafter.

Although the gp120 isolates described herein can be used as a vaccine asdescribed above, the amino acid sequences can also be used alone or incombinations in the same type of formulation for use as an immunogen, toinduce antibodies that recognize the isolate(s) present in theimmunogen. Immunogens are formulated in the same manner as vaccines andcan include the same excipients, etc. Antibodies induced by theimmunogens can be used in a diagnostic to detect the HIV strain in theimmunogen or to affinity purify the strain.

gp120 Polypeptide Sequences and Chemokine Receptors

While CD4 is the primary cellular receptor for HIV-1, it is notsufficient for entry of HIV-1 into cells. Co-receptors required inconjunction with CD4 have been identified. These co-receptors aremembers of the chemokine receptor family of seven-transmembraneG-protein coupled receptors. The chemokine superfamily is subdividedinto two groups based on the amino terminal cysteine spacing. The CXCchemokines are primarily involved in neutrophil-mediated inflammation,and the CC chemokines tend to be involved in chronic inflammation. Atleast five CC chemokine receptors, designated CC-CKR1–5 (also known inthe art as CCR1–5), and at least four CXC chemokine receptors,designated CXC-CKR1–4 (also known as CXCR-1–4), have been identified.

CXC-CKR-4 (CXCR-4), which has also been called the alpha-chemokinereceptor fusin, serves as an entry cofactor for T-cell-tropic HIV-1strains. CC-CKR-5 (CC-RS), which has been called beta-chemokinereceptor, together with its related family members, such as CC-CKR-2band CC-CKR3, serve as entry cofactors for macrophage-tropic HIV-1strains. T-cell-tropic strains can infect primary T-cells and T-celllines, but not macrophages, whereas macrophage-tropic strains can infectmacrophages and primary T-cells, but not T-cell lines. T-cell- andmacrophage-tropic strains are discussed more fully in Deng et. al.,Nature 381:661–666 (1996), which is hereby incorporated by reference inits entirety. Examples of T-cell-tropic strains include laboratoryisolates, such as IIIB and MN. Macrophage-tropic strains include primaryisolates, including but not limited to CM244, GNE6, GNE8, andbreakthrough viruses from vaccinees immunized with gp120-based vaccines.Dual-tropic strains can, use both types of co-receptors, entering cellsvia CXC-CKR-4 or via one or more CC-CKR family members, preferablyCC-CKR-5, CC-CKR-2b, or CC-CKR-3. While the present invention is notintended to be bound or limited by any one theory, the entry of T-celltropic and macrophage-tropic HIV-1 strains is believed to provide aunifying explanation of the differences in cell tropism between viralstrains, the resistance to HIV-1 infection by many CD4-transfectednonprimate cells, and the HIV-1-infection resistance of a portion of thehuman population.

Accordingly, in one embodiment is a vaccine containing (1) a first gp120polypeptide sequence, or fragment thereof, from a macrophage-tropicHIV-1 strain and/or a second gp120 polypeptide sequence, or fragmentthereof, from a T-cell tropic strain, in combination with (2) abreakthrough isolate HIV gp120 polypeptide sequence, or fragmentthereof, from a vaccinee vaccinated with the first and/or second HIVgp120 polypeptide sequence. Preferably, the vaccine includes at leasttwo gp120 polypeptide sequences that bind to different chemokinereceptors. In one embodiment, the vaccine includes first and secondgp120 polypeptide sequences that bind to different chemokine receptors.In addition, the breakthrough isolate gp120 polypeptide sequence canbind to a different chemokine receptor than the chemokine receptor(s)bound by either or both of the first and second gp120 polypeptidesequence(s).

A preferred T-cell tropic strain is a laboratory isolate, mostpreferably MN. Preferred macrophage-tropic viruses for use in theinvention are GNE6 and GNE8, which are representative of thebreakthrough viruses disclosed herein and differ from MN in that theirgp120s induce the formation of antibodies that recognize the gp120sequences (e.g., the V3 domain) involved in binding to CC chemokinereceptors, such as CXC-CKR-5.

In one embodiment, HIV infection is prevented by administering one ormore chemokine receptor-binding gp120 polypeptide sequences, orfragment(s) thereof containing appropriate chemokine receptor-bindingdomains, in a vaccine, such as those described above. Preferably, thevaccine also includes one or more CD4-binding gp120 polypeptidesequences or appropriate fragments thereof. Such vaccines induceanti-HIV antibodies that inhibit viral gp120-chemokine receptor or -CD4binding. In addition, such gp120 polypeptides can directly inhibit HIVinfection by binding to one or more co-receptors for HIV infection, suchas CD4 or a chemokine receptor, thus providing a prophylactic ortherapeutic effect in treating HIV infection. Preferably, gp120polypeptide sequences useful in this regard contain the T-cell binding(TCB) domain.

Various uses of chemokine receptor-binding gp120 polypeptides arediscussed below with regard to the CC chemokine receptor family.However, those skilled in the art recognize that this discussion appliesequally to CXC chemokine receptors that act as cofactors in HIVinfection.

The gp120 polypeptides can be used as a composition containing one ormore gp120 polypeptides, as described for use as a vaccine or immunogen.The composition can be administered, prophylactically ortherapeutically, to a patient at risk of infection or in need of suchtreatment using the dosages and routes and means of administrationdescribed herein. However, chronic administration may be preferred anddosages can be adjusted accordingly. It is noted that in vivoadministration can also induce antibodies that bind viral gp120, furtherinhibiting virus binding to CC-CKR.

The gp120 polypeptides can also be used in screening assays to identifyantagonists of CC-CKR. For example, candidate antagonists can bescreened for inhibition of binding of gp120 to a CC-CKR CC-CKR receptorthat is isolated and attached to a surface (e.g., plastic dish) orrecombinantly or naturally expressed on the surface of a cell.Antagonists can either bind gp120 or bind receptor. Preferred candidateantagonists include gp120 compounds, small gp120 peptides (5 to 20 aminoacids in length, preferably 7 to 10 amino acids in length) orpeptidomimetics of gp120 that bind receptor, monoclonal antibodies thatbind gp120, and small organic molecules that bind either gp120 orreceptor.

The antibodies induced by the gp120 polypeptides can also be used toinduce anti-idiotype antibodies that bind CC chemokines. Theseanti-idiotype antibodies can be screened for binding to an anti-gp120polypeptide antibody and inhibiting gp120 from binding CC-CKR receptor.Such anti-idiotype antibodies mimic gp120 by binding to CC-CKR receptor.Such antibodies, preferably human antibodies, can be obtained in anumber of ways, such as human antibodies from combinatorial libraries(e.g., Burton et al. Adv. Immunolo. (1994) 57:191–280). It is nowpossible to produce transgenic animals (e.g., mice) that are capable,upon immunization, of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example,homozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice results in theproduction of human antibodies upon antigen challenge as described inJakobovitis et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993);Jakobovits et al., Nature 362:255–258 (1993); Bruggermann et al., Yearin Immuno. 7: 33 (1993).

Alternatively, phage display technology as described by McCafferty etal., Nature 348:552–553 (1990) can be used to produce human antibodiesand antibody fragments in vitro from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are closed in-frame either into either a majoror minor coat protein gene of a filamentous bacteriophage, such as M13or fd, and displayed as functional antibody fragments on the surface ofthe phage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Phage displaycan be performed in a variety of formats as reviewed by, for example,Johnson, et al., Current Opinion in Structural Biology 3:564–571 (1993).

Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352: 624–628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors (or embryonic cells) can beconstructed. It has been demonstrated that antibodies to a diverse arrayof antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Marks et al., J. Mol. Biol., 222:581–597 (1991), or Griffith et al., EMBO J., 12: 725–734 (1993).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (Marks et al.,Bio/Technol. 10:779–783 [1992]). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., Nucl. Acids Res., 21: 2265–2266(1993).

Accordingly, antibodies that bind CC-CKR can be obtained by screeningantibodies or fragments thereof expressed on the surface ofbacteriophage in combinatorial libraries or in other systems asdescribed above with a gp120 monoclonal antibody that inhibits gp120binding to receptor.

In addition to screening antibodies with a gp-120 antibody, random orcombinatorial peptide libraries can be screened with either a gp120antibody or the gp120 compounds of the invention. Approaches areavailable for identifying peptide ligands from libraries that compriselarge collections of peptides, ranging from 1 million to 1 billiondifference sequences, which can be screened using monoclonal antibodiesor target molecules. The power of this technology stems from thechemical diversity of the amino acids coupled with the large number ofsequences in a library. See for example, Scott et al., Cur. OpenBiotechnol. 5(1):40–8 (1994); Kenan et al. Trends Biochem. Sci. (1994)19(2):57–64. Accordingly, the monoclonal antibodies, preferably humanmonoclonals or fragments thereof, generated as discussed herein, finduse in treatment by inhibiting or treating HIV infection or diseaseprogression, as well as in screening assays to identify additionalpharmaceuticals.

Production of gp120

gp120 for a vaccine can be produced by any suitable means, as with priorart HIV gp120 subunit vaccines. Recombinantly-produced or chemicallysynthesized gp120 is preferable to gp120 isolated directly from HIV forsafety reasons. Methods for recombinant production of gp120 aredescribed below.

Oligonucleotides encoding gp120 from breakthrough isolates and capableof expressing gp120 can be prepared by conventional means. For example,the nucleotide sequence can be synthesized. Alternatively, another HIVnucleotide sequence encoding gp120 can be used as a backbone and alteredat any differing residues as by site-directed mutagenesis. Site-directedmutagenesis is described in Kunkel et al, Proc. Natl. Acad. Sci. (USA)82:488–492 (1985) and Zoller et al, Nuc. Acids Res. 10:6487–6500 (1982)and is well known.

In a preferred embodiment, the nucleotide sequence is present in anexpression construct containing DNA encoding gp120 under thetranscriptional and translational control of a promoter for expressionof the encoded protein. The promoter can be a eukaryotic promoter forexpression in a mammalian cell. In cases where one wishes to expand thepromoter or produce gp120 in a prokaryotic host, the promoter can be aprokaryotic promoter. Usually a strong promoter is employed to providehigh-level transcription and expression.

The expression construct can be part of a vector capable of stableextrachromosomal maintenance in an appropriate cellular host or may beintegrated into host genomes. Normally, markers are provided with theexpression construct which allow for selection of a host containing theconstruct. The marker can be on the same or a different DNA molecule,desirably, the same DNA molecule.

The expression construct can be joined to a replication systemrecognized by the intended host cell. Various replication systemsinclude viral replication systems such as those from retroviruses,simian virus, bovine papilloma virus, or the like. In addition, theconstruct may be joined to an amplifiable gene, e.g. the DHFR gene, sothat multiple copies of the gp120 DNA can be made. Introduction of theconstruct into the host will vary depending on the construct and can beachieved by any convenient means. A wide variety of prokaryotic andeukaryotic hosts can be employed for expression of the proteins.

Preferably, the gp120 is expressed in mammalian cells that provide thesame glycosylation and disulfide bonds as in native gp120. Expression ofgp120 and fragments of gp120 in mammalian cells as fusion proteinsincorporating N-terminal sequences of Herpes Simplex Virus Type 1(HSV-1) glycoprotein D (gD-1) is described in Lasky, L. A. et al., 1986(Neutralization of the AIDS retrovirus by antibodies to a recombinantenvelope glycoprotein) Science 233: 209–212 and Haffar, O. K. et al.,1991 (The cytoplasmic tail of HIV-1 gp160 contains regions thatassociate with cellular membranes.) Virol. 180:439–441, respectively. Apreferred method for expressing gp120 is described in the examples. Inthe examples, a heterologous signal sequence was used for convenientexpression of the protein. However, the protein can also be expressedusing the native signal sequence.

An isolated, purified gp120 polypeptide having one of the amino acidsequences illustrated in Table 1 can be produced by conventionalmethods. For example, the proteins can be chemically synthesized. In apreferred embodiment, the proteins are expressed in mammalian cellsusing an expression construct of this invention. The expressed proteinscan be purified by conventional means. A preferred purificationprocedure is described in the examples.

gp120 Fragments

The present invention also provides gp120 fragments that are suitablefor use in inducing antibodies for use in a vaccine formulation. Atruncated gp120 sequence, as used herein, is a fragment of gp120 that isfree from a portion of the intact gp120 sequence beginning at either theamino or carboxy terminus of gp120. A truncated gp120 sequence of thisinvention is free from the Cs domain. The CS domain of gp120 is a majorimmunogenic site of the molecule. However, antibodies to the region donot neutralize virus. Therefore, elimination of this portion of gp120from immunogens used to induce antibodies for serotyping isadvantageous.

In another embodiment, the truncated gp120 sequence is additionally freefrom the carboxy terminal region through about amino acid residue 453 ofthe gp120 V5 domain. The portion of the V5 domain remaining in thesequence provides a convenient restriction site for preparation ofexpression constructs. However, a truncated gp120 sequence that is freefrom the entire gp120 V5 domain is also suitable for use in inducingantibodies.

In addition, portions of the amino terminus of gp120 can also beeliminated from the truncated gp120 sequence. In particular, thetruncated gp120 sequence can be free from the gp120 signal sequence. Thetruncated gp120 sequence can be free from the carboxy terminus throughamino acid residue 111 of the gp120 C1 domain, eliminating most of theC1 domain but preserving a convenient restriction site. However, theportion of the C1 domain through the V2 cysteine residue that forms adisulfide bond can additionally be removed, so that the truncated gp120sequence is free from the carboxy terminus through amino acid residue117 of the gp120 C1 domain. In a preferred embodiment, the truncatedgp120 sequence is free from the amino terminus of gp120 through residue111 of the C1 domain and residue 453 through the carboxy terminus ofgp120.

The truncated gp120 sequences can be produced by recombinantengineering, as described previously Conveniently, DNA encoding thetruncated gp120 sequence is joined to a heterologous DNA sequenceencoding a signal sequence.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation is within the capabilitiesof one having ordinary skill in the art in light of the teachingscontained herein. Examples of the products of the present invention andrepresentative processes for their isolation, use, and manufactureappear-below, but should not be construed to limit the invention. Allliterature citations herein are expressly incorporated by reference.

EXAMPLES Materials and Methods

Specimen collection from human volunteers. Blood was collected fromMN-rgp120-immunized individuals who were infected with HIV-1 whileparticipating in Phase I (NIH Protocol AVEG 016) and Phase II (NIHProtocol AVEG 201) HIV-1 vaccine trials sponsored by the NationalInstitutes of Health (NIH). The demographics of the subjects in thestudy, and the study design have been described in McElrath; Seminars inCancer Biol. 6:1–11 (1995); McElrath et al.; Abstracts from EighthAnnual Meeting of the National Cooperative Vaccine Development Groupsfor AIDS. Bethseda, Md. 216 (1996). Specimens were obtained according toan informed consent protocol approved by the institutional review boardsof the participating institutions. In the experimental section, the timeof HIV-1 infection is specified with regard to data provided by the NIHAIDS Vaccine Evaluation Network where PCR (RNA) and/or serologic assayswere used to detect HIV-1 infection.

Sample preparation for cloning HIV-1 envelope glycoproteins. Peripheralblood mononuclear cells (PBMCS) from HIV-1 infected vaccinees wereprepared from heparinized venous blood by FICOLL-HYPAQUE gradientcentrifugation. Cell number and viability were determined. Afterseparation, PBMCs were washed twice in phosphate-buffered saline andsuspended at a cell density of 6×10⁶ cells/ml in PCR lysis buffer (50 mMKCl, 10 mM Tris (pH 8.4), 2.5 mM MgCl₂, 0.1 mg/ml gelatin (Sigma), 0.45%NONIDET P40 detergent, 0.45% TWEEN 20 detergent (both detergents arecommercially available from United States Biochemical Corp.) and 0.06mg/ml Proteinase K (Gibco BRL) to lyse the cells. The lysate wasincubated at 50–60° C. for 1 hour, followed by inactivation of theProteinase K at 95° C. for 10 minutes. Lysates were shipped frozen andstored at −70° C. until use.

Polymerase chain reaction (PCR) amplification. Samples were subjected totwo rounds of PCR amplification using the nested primers describedbelow. In the first round, 25 μl aliquots of PBMC lysates (containingabout 1 μg genomic DNA) were mixed with an equal volume of a PCRreaction mix containing 400 μM each dNTP, 200 μg/ml BSA (Sigma ChemicalCorporation, RIA grade) and about 100 pmoles of each primer in 50 mMKCl, 20 mM Tris (pH 8.4) and 3 mM MgCl₂. After an initial 10 minutedenaturation step at 95° C., 5 units of Taq polymerase (AMPLITAQ, PerkinElmer Cetus) were added during an 55° C. soak step, and samples wereoverlayed with mineral oil.

The PCR profile was as follows: 2 cycles having 1 minute at 55° C., 2.5minutes at 72° C. and 1 minute at 94° C., followed by 28 cycles with 30seconds at 55° C., 2.5 minutes at 72° C. and 45 seconds at 94° C., andan extension step at 72° C. for 5 minutes.

Aliquots of 10 .mu.l from the first-round reactions were re-amplifiedwith appropriate nested primers in a final reaction volume of 100 .mu.l,using either the reagents and profile described above or the reagentsand profile described in the PCR Optimizer Kit (Invitrogen.) PCRreaction products were purified using QIAQUICK-spin columns (QiagenInc.) The primer pair used in the first round was either 120.os.F(5′-gggaattcggatccAGAGCAGAAGACAGTGGCAATGA with homologous sequence atposition 6248–6270 of HIVPV22) (SEQ. ID. NO: 47) or JM11A(5′-ctcgag-CTCCTGAAGACAGTCAGACTCATCAAG at position 6048–6074) (SEQ. ID.NO: 48) in the forward direction [Kusumi et al.; J. Virol. 66:875(1992)] combined with 120.os.R(5′-ggtctagaagctttaGCCCATAGTGCTCCTGCTGCT-CC at position 7836–7859) (SEQ.ID. NO: 49) in the reverse direction. The internal nested primers were120.BX.F (5′-gggcggatcctcgaGGTACCTGTRTGGAAAG-AAGCA at position6389–6410; R: A or G) (SEQ. ID. NO: 50) and 120.is.R(5′-ggtctagaagctttaTGCTCCYAAGAACCCAAGGAACA at position 7819–7841; Y: Tor C) (SEQ. ID. NO: 51). Heterologous primer sequences are shown inlower case letters.

Subcloning of PCR products and the expression of recombinant envelopeglycoproteins as fusion proteins. The HIV-1 envelope glycoprotein gp120sequences were cloned and expressed as chimeric genes and fusionproteins, where the signal sequence and 27 amino acids from the mature Nterminus of herpes simplex virus type 1 (HSV-1) were fused to theN-terminal sequences of the gp120 genes, corresponding to amino acid 13of the mature gp120 sequence. PCR products containing gp120 sequencesfrom the breakthrough specimens were cloned into pRK5 expression plasmidas chimeric genes using combinations of restrictions sites engineeredinto the heterologous PCR primer tails and the Xho I site engineeredinto the N-terminal sequence of HSV-1 gD.

The resulting double-stranded DNA was sequenced with Sequenase and thedGTP Reagent Kit (United States Biochemical Corp.). Sequences fromglycoprotein D were provided to enhance expression and to provide a flagepitope to facilitate protein analysis, as described in Berman et al.;J. Virol. 7:4464–9 (1992); Nakamura et al.; AIDS and Human Retroviruses8:1875–85 (1992); and Nakamura et al.; J. Virol. 67:6179–91 (1993).

Briefly, isolated DNA fragments generated by the PCR reaction wereligated into a plasmid (pRK.gD-5, pRKgDstop) designed to fuse the gp120fragments, in frame, to the 5′ sequences of the glycoprotein D (gD) geneof Type 1 Herpes Simplex Virus (gD-1) and the 3′ end to translationalstop codons. The fragment of the gD-1 gene encoded the signal sequenceand 25 amino acids of the mature form of HSV-1 protein. To allow forexpression in mammalian cells, chimeric genes fragments were cloned intothe pRK5 expression plasmid (Eaton et al., Biochemistry 291:8343–8347(1986)) that contained a polylinker with cloning sites and translationalstop codons located between a cytomegalovirus promotor and a simianvirus 40 virus polyadenylation site.

The resulting plasmids were transfected into the 293s embryonic humankidney cell line (Graham et al., J. Gen. Virol. 36:59–77 (1977)) using acalcium phosphate technique (Graham et al., Virology 52:456–467 (1973)).Growth conditioned cell culture media was collected 48 hr aftertransfection, and the soluble proteins were detected by ELISA or byspecific radioimmunoprecipitation where metabolically labeled proteinsfrom cell culture supernatants were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (PAGE) and visualized byautoradiography as described in Berman et al., J. Virol. 63:3489–3498(1989) and Laemmli Nature 227:680–685 (1970).

Serologic assays. Sera were assayed for antibodies to rgp120, antibodiesto synthetic gp120 V3 domain peptides corresponding to sequences fromthe gp120 V3 domain, and antibodies able to inhibit the binding ofMN-rgp120 to cell surface CD4 using serologic assays described in Bermanet al.; J. Virol. 7:4464–9 (1992); Nakamura et al.; AIDS and HumanRetroviruses 8:1875–85 (1992); and Nakamura et al.; J. Virol. 67:6179–91(1993). Endpoint titers of antibody binding to gp120 and V3 peptideswere determined using three fold-serial dilutions of sera. The endpointdilution titer was defined as the last dilution that produced an opticaldensity value that was two times higher than the mean of the opticaldensities of 1:50 diluted, pooled, normal human sera. Antibody titerswere calculated by a computer program that interpolated values betweenantibody dilutions. The inter-assay coefficient of variation of positivecontrol standard sera was 35%.

Binding of monoclonal antibodies to rgp120 from breakthrough viruses. AnELISA similar to that described by Moore et al.; AIDS 3:155–63 (1989)was used to measure the binding of various monoclonal antibodies (MAbs)to rgp120s from breakthrough viruses. Briefly, Nunc-Immuno plates(Maxisorp, certified) were coated (100 μl at 5 μg/ml in PBS at 4° C.overnight) with an affinity-purified sheep polyclonal antiserum to apeptide at the C terminus of gp120 (D7324, International Enzymes,Fallbrook, Calif.). After washing once with PBS-0.05% TWEEN-20detergent, the plates were blocked with PBS-1.0% BSA for 30–60 minutesat room temperature. Cell culture supernatants from 293s cells, dilutedto contain equivalent amounts of the gD-rgp120 fusion protein, wereadded and incubated for 2 hours at room temperature followed by threewashes with PBS-0.05% TWEEN-20 detergent. Various MAbs were diluted inPBS-1.0% BSA and 100 μL of the diluted MAbs were added to each well andincubated for 1 hour at room temperature.

The plates were washed 3 times and incubated with 100 μl of ahorseradish peroxidase-conjugated second antibody (goat anti-mouse oranti-human IgG, Cappel) for 1 hour at room temperature. After 3 washesthe plates were developed and the OD₄₉₂ (optical density at 492 nm) readin a plate reader. Growth conditioned cell culture supernatants werenormalized by dilution based on binding by MAb 5B6 which is specific forHSV-1 glycoprotein D fusion protein.

Virus neutralization assays. The ability of vaccinee sera to inhibitinfection of MT4 cells by HIV-1_(MN) was measured in a cytopathicityassay where cell viability was quantitated using a calorimetricindicator dye, as described in Robertson et al.; J. Virol. Methods20:195–202 (1988). Briefly, a virus stock of HIV-1_(MN) (obtained fromDr. Michael Norcross, U.S. Food and Drug Administration) was prepared asthe clarified supernatant from chronically infected H9/HIV-1_(MN) cellculture. H9 cells chronically infected with HIV-MN were pelleted andresuspended in one-tenth the original volume of medium. Cell-associatedvirus was released by the mechanical shearing effects of rapid vortexingof the cells as described in Wrin et al.; J. Virol. 69:39–48 (1995).

An amount of virus sufficient to ensure complete cell lysis killing in 7days was incubated with three-fold serial dilutions of test antisera,and then used to challenge MT4 T-lymphoid cells in 10% FCS/RPMI-1640cell culture media. The cultures were incubated for 7 days at 37° C. in5% CO₂, and then cell viability was tested by the dye MTT, as describedby Robertson et al.; J. Virol. Methods 20:195–202 (1988). Virusneutralization endpoints were quantitated by measurement of OD at570–650 nm, and then the endpoint titers were calculated as thereciprocal of the antiserum dilution giving a signal that was two-foldabove the control signal with unprotected (killed) cells. These titerswere typically twice those calculated at 50% protection.

Results

Immunization history of infected subjects. Since 1992, 499 adults havebeen immunized with MN-rgp120 in Phase I trials in low or moderate riskindividuals and in a Phase II clinical trial involving moderate to highrisk individuals. The studies described herein entail the genetic andimmunologic characterization of the first seven of nine individuals whobecame infected with HIV-1 through high risk behavior during the courseof these trials. A listing of the trials and summary of the status ofthe vaccinees is presented in Table 2A. A listing of the analysis of thevaccinees is presented in Table 2B.

TABLE 2A Description of Vaccinees Infected with HIV-1 After Immunizationwith MN-rgp120 ‡Antigen dose/ Study No. Case No. *Risk Group Adjuvant016 C6 M/H 300/QS21 016 C8 M/H 600/QS21 016 C15 M/H 300/QS21 201 C7 M/H600/Alum 201 C11 M/H 600/Alum 201 C10 M/IDU 600/Alum 201 C17 M/IDU600/Alum *M/H indicates male homosexual; M/IDU indicate male intravenousdrug user. ‡numbers indicate dose in micrograms of MN-rgp120 injectedper immunization; QS21 indicates antigen was formulated in QS21adjuvant; Alum indicates MN-rgp120 formulated in aluminum hydroxide.

TABLE 2B Description of Vaccinees Infected with HIV-1 After Immunizationwith MN-rgp120 Injection Injections Time of

Interval: Case Schedule before HIV-1+ to HIV-1+ No. (months) HIV-1+(months) (months) C6 0, 1, 10.5 2 4.00 2.00 C8 0, 1 2 4.00 3.00 C15 0,1, 2 3 6.25 4.00 C7 0, 1, 6, 12 3 9.25 3.00 C11 0, 1, 6, 12 4 19.50 6.75C10 0, 1, 6, 19 3 19.50 13.50 C17 0, 1, 6, 18 4 24.75 6.25

indicates interval between last immunization and detection of HIV-1infection.

Three of the infections occurred in a Phase I trial (NIH Protocol AVEG201) that compared the safety and immunogenicity of MN-rgp120 formulatedin two different adjuvants (alum and QS21), and four of the infectionsoccurred in a Phase II trial aimed at establishing the safety andimmunogenicity of MN-rgp120 in various high risk groups (e.g.,intravenous drug users, homosexual and bisexual males, and partners ofHIV-1 infected individuals).

Of the seven infections studied (Table 3), two (C6 and C8) occurredafter two injections, three (C7, C10 and C15) occurred after threeinjections, and two (C11 and C17) occurred after receiving the fourscheduled injections. The interval between receiving the lastimmunization and becoming infected was 2 to 13.5 months.

TABLE 3 Peak Post Boost MN-rgp120 Antibody Titers in Vaccinees thatBecame Infected with HIV-1 Injections C6 C8 C15 C7 C11 C10 C17 1 <502185 79 <50 1890 na na 2 21539 10125 na 413 32696 7771 7056 3 # # 44609707 34728 11627 18413 4 # # # # # # 11340 # indicates specimen notanalyzed because of HIV-1 infection. na indicates the sample was notavailable for testing. boldface indicates unusually low antibody titers.

Antibody response to gp120 in vaccinated individuals. The magnitude andspecificity of the antibody response to MN-rgp120 was measured by ELISAin all infected individuals throughout the course of the immunizationregime (FIG. 1). Five of the seven subjects exhibited normal antibodyresponse kinetics that included a small but reproducible primaryresponse (1:100–1:2,000) and a strong secondary (booster) response(titters ranging from 1:7,000–1:32,000), and antibody responsesfollowing third and fourth injections that were similar or marginallyhigher than those achieved after the second immunization (FIG. 1, Table3).

The antibody response observed in C7 (FIG. 1C) was unusual in that noantibodies were detectable after the primary injection and a titer ofonly 1:350 was detected after the second injection. It thus appearedthat C7 did not respond to the primary immunization, and that theantibody response obtained after the second injection represented aprimary immune response. Consistent with this hypothesis, the thirdinjection elicited a titer of only 1:9,707, typical of those normallyseen after two immunizations.

An a typical antibody response was also seen in subject C15 (FIG. 1G)who was immunized according to an accelerated immunization schedule of0, 1, and 2 months. As expected, the antibody titer seen in this subject(1:4,460) was at the low end of what is typically achieved after twoimmunizations and was far below normal values for three immunizations.The lack of an effective booster response after the third immunizationof C15 was not surprising in view of previous studies where anaccelerated 0, 1, and 2 month immunization schedule in baboons [Andersonet al.; J. Infect. Dis. 160:960–9 ((1989)] similarly prolonged thesecondary response and failed to elicit an effective tertiary boosterresponse.

Retrospective analysis of serum and plasma from subjects C6 (FIG. 1A)and C8 (FIG. 1B) indicated that they became infected with HIV-1 at somepoint between the second and third immunizations. Serologic evidence ofHIV-1 infection was evident in the gp120 antibody assays where thetiters failed to decline two weeks after the second injection andinstead formed an uncharacteristic high titer plateau (FIGS. 1A and 1B).A similar plateau in MN-rgp120 titer after the third injection,suggested that subject C7 became infected around week 36, approximately16 weeks after receiving the third injection (FIG. 1C). Subjects C10(FIG. 1E), C11 (FIG. 1D), C15 (FIG. 1G), and C17 (FIG. 1F) developedunexpected increases in gp120 titers, typical of HIV-1 infection, aftereither the third or fourth immunizations. The data obtained demonstratethat immunologic priming for MN-rgp120 antibody responses isinsufficient to provide universal protection from HIV-1 infection.

Antibody titers to the V3 domain. To further characterize the antibodyresponse to gp120, antibody titers were measured to a synthetic V3domain peptide of MN-rgp120 containing the principal neutralizingdeterminant (PND). Five of the seven subjects developed good V3 titers(1:400 to 1:4000) after the second immunization, however two subjects(C7 and C15) required three immunizations before developing significanttiers (FIGS. 1C and 1G). As had been observed previously (11), the peakV3 titers in some individuals (e.g. C11, C10, C17) appeared to declinewith each successive immunization (FIGS. 1D, 1E, and 1F). After HIV-1infection, two patterns of V3 reactivity were observed. Three subjects(C6, C7, and C10) showed large increases in titer to V3 domain peptides(FIGS. 1A, 1C, and 1E) whereas C8 (FIG. 1B) showed a large decrease inV3 titer. At the time of analysis, the data were insufficient to drawany conclusions regarding the changes in V3 titers in response to HIV-1infection in subjects C11, C15 and C17.

The results obtained indicate that the ability to form antibodiesreactive with the V3 domain at various time-points prior to HIV-1infection is not a valid correlate of protective immunity against allstrains of HIV-1.

CD4 Inhibition titers. Antibodies that block the binding of gp120 to CD4represent a heterogeneous class of virus neutralizing antibodies. Someare known to bind to the C4 domain of gp120 [Nakamura et al.; J. Virol.67:6179–91 (1993); Anderson et al.; J. Infect. Dis. 160:960–9 ((1989)],and some are known to recognize conformation dependent discontinuousepitopes [Berman et al.; J. Virol. 7:4464–9 (1992); Nakamura et al.; J.Virol. 67:6179–91 (1993); McKeating et al.; AIDS Research and HumanRetroviruses 8:451–9 (1992); Ho et al.; J. Virol. 65:489–93 (1991);Barbas et al.; Proc. Natl. Acad. Sci. USA 91:3809–13 (1994)].

One way to detect antibodies to both types of epitopes is to measure theability of vaccinee sera to prevent the binding of [¹²⁵I]-labeled gp120to cell surface CD4 [[Nakamura et al.; AIDS and Human Retroviruses81875–85 (1992); Nakamura et al.; J. Virol. 67:6179–91 (1993)]. CD4blocking titers were detected in all seven of the vaccinees prior toinfection (FIG. 2) with peak titers that ranged from 1:10–1:300. At thelast time point prior to infection, the CD4 titers in five of the sevenvaccinees was low (1:30 or less). One vaccinee (C17), however, possesseda CD4 blocking titer of about 1:300 prior to infection (FIG. 2F). Thus,the lack of antibodies that block the binding of MN-rgp120 to CD4 cannotaccount for all of the infections. Large increases in CD4 blockingtiters (1:100–1:1,000) were seen in five of the seven subjects afterHIV-1 infection. These included vaccinees C6, C7, C8, C10, and C11.These results demonstrate that the CD4 blocking titers elicited byMN-rgp120 were lower than those elicited by natural infection.

Virus neutralizing activity. The virus neutralizing activity of antiserafrom MN-rgp120-immunized subjects was measured using a calorimetricassay that measured the viability of MT-4 cells after incubation withantibody treated virus (HIV-1_(MN)). Since the actual date of infectionwas not known for any of the breakthrough infections, and serum sampleswere collected infrequently, the magnitude of the neutralizing antibodyresponse at the time of infection is not known for any of the vaccinees.

Of the seven infections examined, the serum sample closest to the timeof infection was that obtained from C7, where a neutralizing titer of1:15 to HIV-1_(MN) was present three weeks prior to detection of HIV-1infection (Table 4). In all other cases, however, the interval betweenthe last injection and the time of infection was 10 to 25 weeks.

TABLE 4 Neutralization Activity of Sera from Vaccinees Infected withHIV-1 Week C6 C8 C15 C7 C11 C10 C17 0  <10*  <10*  <10*  <10*  <10* <10*  <10* 2  <10  <10  <10 — — — — 4  <10*  <10* nd*  <10*  <10*  <10* <10* 6    10    80 —  <10    30   150   150 8 — — nd* — — — — 10 — —   35 — — — — 15 — — —  <10 — — — 16   150#   250# — —    30    10  <1024   150#  <10*    20*  <10*  <10* 26    70   500   200   400 30 — —   40   100 33    15 — — — 35 —   100 — — 36    30# —    10    40 52   30*  <10  <10 54   250 — — 57   100 — — 63    90 — — 64 — —  <10 77   40# — — 78   500#    10* 80   100 84    60 90   150 104   150#*indicates immunization. #indicates HIV-1 positive. nd indicates notdone. — indicates sample not available.

When sera from the two early infections were examined (Table 4), oneindividual (C6) had a peak neutralizing titer of 1:10 ten weeks prior todetection of HIV-1 infection, whereas the other individual (C8) had aneutralizing titer of 1:80 ten weeks prior to detection of HIV-1infection. Subject C15, who was immunized according to an acceleratedimmunization schedule, developed a neutralizing titer of 1:35 after thethird injection, 14 weeks prior to HIV-1 infection. Subject C10, who hada peak neutralizing titer of 1:200 following the third immunization(week 24), had no detectable titer at week 52, six months prior to thefirst indication of HIV-1 infection (week 78).

Subject C11 possessed a neutralizing titer of 1:90 at fourteen weeksprior to detection of HIV-1 and a peak titer of 1:500 following thethird immunization. Similarly vaccinee C17 had a neutralizing titer of1:150 fourteen weeks prior to infection and a peak titer of 1:400 at twoweeks after the third immunization.

Based on the rate of decay of the gp120 response of approximately twomonths [Belshe et al.; JAMA 272(6):475–80 (1994)], as well as theobservation that neutralizing titers of 1:150 decayed to 1:10 in 10weeks in vaccinees C10 and C17, it appears that neutralizing titers inC8, C15, C11, and C17 could have declined to 1:10 or less in theintervals between the last pre-infection serum sample and the time ofHIV-1 detection.

The results of these studies demonstrated that all vaccinees developedsome level of virus-neutralizing antibodies at some time prior to HIV-1infection, and that the magnitude of the neutralizing response wasprobably low at the time of infection. In general, the magnitude of thevirus-neutralizing response observed in the individuals that becameinfected with HIV-1 was comparable to that seen in non-infectedvaccinees as described in Belshe et al.; JAMA 272(6):475–80 (1994).

Sequences of Viruses. To evaluate the similarity of the breakthroughviruses with the vaccine antigen, nucleotide sequences for gp120 fromall seven breakthrough viruses were determined. Envelope glycoproteingenes were amplified from proviral DNA using the polymerase chainreaction. Sequences were obtained by direct amplification of DNA fromlysates of gradient-purified lymphocytes obtained directly from patientblood without any intermediate tissue culture or amplification step.

A listing of the complete gp120 sequences (two clones per specimen) isprovided in FIG. 3. All seven envelope glycoproteins possessed sequencestypical of subtype (lade) B viruses. The overall homology with MN-rgp120ranged from 69–80% (Table 5).

TABLE 5 Comparison of MN-rgp120 Sequence with Sequences from InfectedVaccinees* MN C6.1 C8.3 C7.2 C11.5 C10.5 C17.1 C15.2 MN 100 79 78 70 7569 80 72 C6.1 100 78 70 81 75 90 79 C8.3 100 68 80 76 84 83 C7.2 100 8073 76 73 C11.5 100 75 70 80 C10.5 100 70 72 C17.1 100 87 C15.2 100 *Dataindicate percent identity.Interestingly, a high percentage (four of seven) of the breakthroughviruses differed from MN-rgp120 by 25–30% [Myers et al.; Retrovirusesand AIDS Database, Los Alamos National Laboratory (1992 and 1995)].Historically this degree of sequence variation is typical ofinter-subtype (intra-clade) variation rather than intra-subtypevariation which is expected to be in the 10–20% range [Myers et al.;Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and19950]. Of the viruses with the greatest homology to MN-rgp120, two (C6and C8) occurred as early infections, prior to complete immunization,and one (C17) occurred as a late infection.

Polymorphism in the V3 Domain. Of particular interest were polymorphismsin regions known to contain epitopes recognized by virus neutralizingantibodies. The best characterized neutralizing epitope, the principalneutralizing determinant (PND), occurs at the tip of the V3 loop. Insubtype B viruses, approximately 60% possess the MN serotype-definingsignature sequence, IGPGRAF (SEQ. ID. NO: 52), based on identity withthe prototypic MN strain of HIV-1 [Berman et al.; Virol. 7:4464-9(1992); Myers et al.; Retroviruses and AIDS Database, Los AlamosNational Laboratory (1992 and 1995); La Rosa et al.; Science 249:932–5(1990)].

Three of the viruses (C6, C8, and C17) possessed the MN serotypesignature sequence (FIG. 3). In contrast, four viruses possessedsequences with radical amino acid substitutions in the PND [IGPGRAW (7),LGPGSTF (11), IGPGRVL (10), and IGPGSAF (15)] (SEQ. ID. NOs. 53–56),respectively), and therefore were classified as “non-MN like” viruses.Of note, each of the four “non-MN-like” sequences were rare (Table 6)and were not typical of the most common “non-MN” variants of subtype Bviruses [Myers et al.; Retroviruses and AIDS Database, Los AlamosNational Laboratory (1992 and 1995)].

TABLE 6 Frequency of Polymorphisms at the Principal NeutralizingDeterminant in HIV-1 Infected Individuals Immunized with MN-rgp120* Ob-served Dataset Frequency V3 Sequence Fre- GNE LANL LANL.1 LaRosaSequence n quency (n = 52) (n = 519) (n = 160) (n = 245) GPGRAF 3 0.420.67 0.57 0.66 0.60 GPGRAW 1 0.14 0.03 0.013 0.06 0.010 GPGRVL 1 0.14<0.02 0.004 <0.006 <0.008 GPGSTF** 1 0.14 <0.02 <0.002 <0.006 <0.004GPGSAF 1 0.14 0.02 0.011 <0.006 <0.004 *Data set GNE refers to acollection of 52 independent isolates collected in 1992; dataset LANLrefers to a collection of 519 sequences reported by Myers et al.,Retroviruses and AIDS Database, Los Alamos National Laboratory 1992 and1995; LANL.1 refers to a collection of 160 epidemiologically unlinkedindividuals provided by B. Korber (personal communication) ; dataset LaRosa refers to sequence data reported by La Rosa et al., Science249:932-5 (1990) . **Sequences were not present in the data setsexamined.

The prevalence of viruses with PND sequences matching the breakthroughviruses ranged from a high of 1.3% (C7) to a low of 0.2% (C11) in alisting of 519 subtype B sequences compiled by the Los Alamos NationalLaboratory [Myers et al.; Retroviruses and AIDS Database, Los AlamosNational Laboratory (1992 and 1995)]. Similarly low frequencies wereobserved in three other independently derived data sets (Table 6). Theoccurrence of these sequences did not differ significantly between datasets collected prior to 1985 [La Rosa et al.; Science 249:932–5 (1990)]and data collected 1992, or from a set of 160 epidemiologically unlinkedindividuals (B. Korber, personal communication). All four sets of dataagreed that the prevalence of viruses with MN-like PND sequences was inthe range of 60%. Based on this data, four of the seven breakthroughinfections were determined to be caused by viruses that fell outside ofthe spectrum of viruses that the vaccine was expected to prevent.

Other features of breakthrough virus V3 domains. Like MN-rgp120, the V3domains of all of the breakthrough viruses were 36 amino acids inlength. However, all seven viruses differed from MN-rgp120 with respectto the number of glycosylation sites and with respect to thesyncytium-inducing (SI) signature sequence.

The sequence of MN-rgp120 is somewhat unusual [Myers et al.;Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and1995)] in that it lacks an N-linked glycosylation site at position 306in the V3 domain. The lack of this glycosylation site does not appear tobe antigenically significant since antisera to MN-rgp120 are known toneutralize a variety of viruses (e.g. SF-2, DU6587-5, DU4489-5, CC) thatpossess a glycosylation site at this position [Berman et al.; J. Virol.7:4464–9 (1992)]

In addition, the V3 domain of MN-rgp120 possessed sequence polymorphisms(R at position 311, K at position 324, K at position 328) typical ofsyncytium inducing viruses [Fouchier et al.; J. Virol. 66:3183–87(1992)], whereas all seven breakthrough viruses possessed sequencesassociated with non-syncytium-inducing viruses. Syncytium-inducingviruses have been associated with rapid disease progression [Tersmetteet al.; J. Virol. 62:2026–32 (1988)] and T cell tropism [O'Brien et al.;Nature (London) 348:69–73 (1990); Shioda et al.; Nature (London)349:167–9 (1991)]. To date viruses with these properties have not beenrecovered from any of the MN-rgp120 immunized volunteers.

Polymorphism in the V1, V2 and C4 domains. Previous investigations haveidentified additional neutralizing epitopes in the V1, V2 and C4 domainsof gp120 [Nakamura et al.; J. Virol. 67:6179–91 (1993); McKeating etal.; AIDS Research and Human Retroviruses 8:451–9 (1992); Ho et al.; J.Virol. 65:489–93 (1991); Barbas et al.; Proc. Natl. Acad. Sci. USA91:3809–13 (1994); McKeating et al.; J. Virol. 67:4932–44 (1993); Mooreet al.; J. Virol. 67:6136–6151 (1993); Davis et al.; J. Gen. Virol.74:2609–17 (1993)].

The best characterized of these neutralizing epitopes is in the C4domain which has attracted special attention because antibodies bindingto this area are known to block the binding of gp120 to CD4 [Moore etal.; AIDS 3:155–63 (1989); McKeating et al.; AIDS Research and HumanRetroviruses 8:451–9 (1992)]. Because the epitope is located in aconserved (C) domain, naturally-occurring polymorphism in this region isfar more limited than in other neutralizing epitopes. Nakamura et al.;J. Virol. 67:6179–91 (1993) reported that the binding of a number ofneutralizing MAbs was dependent on K at position 429.

Comparison of the sequence of MN-rgp120 with other strains of HIV-1showed that a common polymorphism, involving the substitution of E forK, occurs at this position. Indeed, substrains of the same virus isolateoften show polymorphism at this position. The HXB2 substrain ofHIV-1_(LAI) contains K at position 429, whereas the BH10, IIIB, and LAVsubstrains of the HIV-1_(LAI) contain E at this position [Nakamura etal.; J. Virol. 67:6179–91 (1993)]. Similarly, the 1984 isolate ofHIV-1_(MN) exhibited E at this position, while the 1990 isolate ofHIV-1_(MN), used to produce MN-rgp120, possessed K at this position.

When the sequences of the infected vaccine recipients were examined(FIG. 3), the virus from subject C17, like MN-rgp120 contained K atposition 429, whereas the six other viruses that differed from thevaccine immunogen possessed E at this position. These resultsdemonstrated that six of the seven breakthrough viruses differed fromthe vaccine immunogen at the CD4-blocking, neutralizing epitope in theC4 domain of gp120.

Studies with monoclonal antibodies have defined neutralizing epitopes inthe V1 and V2 domains of gp120 [McKeating et al.; J. Virol. 67:4932–44(1993); Moore et al.; J. Virol. 67:6136–6151 (1993); Davis et al.; J.Gen. Virol. 74:2609–17 (1993)]. Like the polymorphisms that occur in theC4 domain, the V2 domains exhibit several common polymorphisms thataffect the binding of virus neutralizing antibodies. One suchpolymorphism occurs at position 171 which is critically important forthe binding of murine MAb 1025, whereas residue 187 is important for thebinding of MAb several MAbs represented by 1088.

When the V2 domain sequences were examined (FIG. 3), all of theinfected-vaccinee viruses differed from MN-rgp120 in that R replaced Gat position 171 and I or V replaced E at position 187. Antibodiesrecognizing these adjacent sites in the V2 domain of MN-rgp120 would notbe expected to neutralize viruses with radical amino acid substitutionsat these position. Thus, all seven breakthrough viruses differed fromMN-rgp120 at a neutralizing epitope in the V2 domain of gp120.

Other neutralizing epitopes have been reported in the V1 domain of gp120[O'Brien et al.; Nature (London) 348:69–73 (1990); McKeating et al.; J.Virol. 67:4932–44 (1993)]. Although the neutralizing epitopes in the V1domain of MN-rgp120 have not been characterized, the polymorphism seenamong the breakthrough viruses in this region was interesting.Particularly striking (FIG. 3) was that the length of this domain rangedfrom 20 amino acids (C17) to 45 amino acids (C6), and the number ofN-linked glycosylation sites ranged from 2 to 6. In contrast, the Vidomain of MN-rgp120 is 31 amino acids in length and encodes threeN-linked glycosylation sites.

Although examination of sequence databases suggest that variation in theV2 region is comparable to the V1 region, the V2 region of thebreakthrough viruses showed less variation than expected. Specifically,the length of the V2 region ranged from 36 amino acids (C7) to 39 aminoacids in length, with six of seven viruses containing three N-linkedglycosylation sites in this domain. A high degree of polymorphism wasfound in the V4 region where sequences ranged from 26 (C10) to 33 (C15,C7) amino acids in length and contained either 4 or 5 N-linkedglycosylation sites.

Antigenicity of envelope glycoproteins from breakthrough viruses. Todetermine the significance of sequence variation on glycoproteinantigenicity, recombinant gp120 was prepared from the viruses of allseven infected vaccinees (FIG. 4). In these studies a series of MAbs wasassembled and their binding to MN-rgp120 was compared to that of rgp120from the vaccinee isolates by ELISA (Table 7).

TABLE 7 Relative Reactivity* of MAb Binding to rgp120 from InfectedSubjects Compared with Binding to MN-rgp120 V3 Discontinuous C8 V2 gp1201034 50.1 1.5E 1025 1024 1088 MN 1.0 1.00 1.00 1.00 1.00 1.00 C6.1 0.370.37 0.17 0.00 0.00 0.00 C6.5 0.33 0.33 0.75 0.00 0.00 0.00 C8.3 0.110.37 0.38 0.00 0.00 0.00 C8.6 0.14 0.34 0.29 0.00 0.00 0.00 C7.2 0.470.60 0.71 0.00 0.00 0.00 C11.5 0.00 0.00 0.17 0.00 0.00 0.00 C11.7 0.000.00 0.17 0.00 0.00 0.00 C10.5 0.33 0.40 0.46 0.24 0.03 0.04 C10.7 0.420.48 0.50 0.29 0.07 0.09 C17.1 0.33 0.52 0.33 0.00 0.30 0.07 C17.3 0.370.56 0.33 0.00 0.38 0.06 C15.2 0.00 0.47 0.92 0.00 0.00 0.00 C15.3 0.000.37 0.63 0.00 0.00 0.00 *Relative reactivity values represent ratio ofoptical densities obtained with rgp120 from patient isolates divided byoptical density obtained for MN-rgp120 at a MAb concentration of 2micrograms per milliliter.

In control experiments, the binding of MAb 5B6 (which is specific forthe HSV gD-1 flag epitope fused to the N terminus of all of the rgp120protein) was used to standardize the amount of gp120 from each isolate(FIG. 5A). These studies demonstrated that the assay was carried outunder conditions where equivalent amount of rgp120s were captured ontowells of microtiter plates.

The antigenic structure of the V3 domain was examined using the 1034 MAb(isolated from mice immunized with MN-rgp120 as described in Nakamura etal.; J. Virol. 67:6179–91 (1993) and the 50.1 MAb (prepared from miceimmunized with a synthetic V3 domain peptide as described in Rini etal.; Proc. Nati. Acad. Sd. USA 90:6325–9 (1993). Both MAbs are known toexhibit potent virus neutralizing activity. When binding to therecombinant proteins was examined, the MAb binding to MN-rgp120 was atleast 10-fold greater than to any of the breakthrough virus envelopeproteins (FIGS. 5B and C). Surprisingly, rgp120 from the three patientisolates (C8, C6, and C17) that possessed the MN serotype-definingsequence, IGPGRAF (SEQ. ID. NO: 52), varied from one another in theirMAb binding activity. Thus, the binding of MAb 1034 and MAb 50.1 torgp120 from C17 was significantly greater than the binding to rgp20sfrom C6 and C8.

A distinction in the epitopes recognized by these MAbs was evident sinceC6-rgp120 and C8-rgp120 gave comparable binding with 50.1, whereas 1034bound better to the C6-derived protein than the C8-derived protein. Thepoorest MAb reactivity was with rgpl20s from C11 and C15. This resultwas consistent with sequence analysis demonstrating that these twoviruses both possessed the radical substitution of S for R at position18 in the V3 domain. Surprisingly, both of these MAbs exhibited betterthan expected binding to rgp120 from the C7 and C10 viruses. LikeMN-rgp120, both proteins contained the penta-peptide, IGPGR sequence(SEQ. ID. NO: 57) in the V3 loop, but differed from MN-rgp120 in that Vand L replaced A and F at positions 319 and 320 in gp120 from C10, and Wreplaced F at position 320 in gp120 from C7. These results indicate thatR at position 318 is essential for the binding of these two MAbs, andthat the epitopes recognized by 1034 and 50.1 are not completelydestroyed by the hydrophobic substitutions at positions 319 and 320.

As predicted from the sequence data, there was little if any binding tothe breakthrough virus rgp120s using MAbs (1088 and 1025) directed tothe V2 region of MN-rgp120 Also consistent with sequence data was theobservation that MAb 1024 directed to the C4 domain of MN-rgp120 gavesome reactivity with C17-rgp120 which, like MN-rgp120 contained K atposition 429, but gave no reactivity with the other isolates thatcontained E at residue 429.

Together, these studies demonstrated that the antigenic structure of allseven breakthrough viruses differed from the vaccine immunogen at threewell characterized neutralizing epitopes.

A totally different pattern of reactivity was observed with the humanhybridoma, MAb 15e, prepared from an HIV-1 infected individual asdescribed in Ho et al.; J. Virol. 65:489–93 (1991). With this MAb, thegreatest binding was achieved with MN-rgp120 and rgp120 from C7, and thepoorest reactivity was seen with the two clones of rgp120 from the C11.Moderate, but comparable reactivity was seen with rgp120s from the C10and C17.

These results demonstrate that the 15e epitope is polymorphic, and thatthe epitope is conserved on MN-rgp120 and rgp120 from C7, but has beenlost on rgp120s from C11. Interestingly, the two different clones ofgp120 derived from C6 gave strikingly different patterns of antibodybinding. Thus, rgp120 from clone C6.5 exhibited strong reactivity withthis antibody, whereas rgp120 from clones C6.1 exhibited significantlyweaker activity with this MAb. Comparison of sequence data (FIG. 3)showed that the two C6 clones differed at 6 amino acid positions. Basedon comparative binding to the other viral proteins of known sequence, itappeared that the substitution of K for I at position 351 in the C3domain of gp120 could account for the difference in binding activity.This result is also consistent with both clones of C11 similarlycontaining a positively-charged K at this position, and also beingpoorly reactive with this MAb. Alternatively, a T for I substitution atposition 439 in the C4 domain could account for the difference in 15ebinding between C6.1 and C6.5. Although the inability of the two C11clones to bind 15e cannot be explained by polymorphism at this positionin the C4 domain, they could be affected by the adjacent T for Msubstitution at position 434.

Discussion

In these studies, the viruses and immune responses in seven of ninevaccinees who became infected with HIV-1 vaccine through high riskactivity while participating in Phase I or Phase 2 trials of MN-rgp120,a candidate HIV-1 vaccine were analyzed. Such infections would beexpected to occur for one of two reasons: 1) lack of sufficient immuneresponse at the time of infection; or 2) infection with viruses thatfall outiside of the antigenic spectrum expected to be covered by thevaccine immunogen. The data indicate that both explanations may beinvolved with the infections observed (Table 8).

TABLE 8 Summary of Breakthrough Infections MN-rgp120 Homoloqous toMN-rgp120 Adequate Homology V3 C4 V2 Case No. Immunization (%) PNDEpitope Epitope C6 − 79 + − − C8 − 78 + − − C15 − 72 − − − C7 − 70 − − −C11 + 75 − − − C10 + 69 − − − C17 + 80 + + −

Two of the infections occurred in individuals who failed to receive theminimum three doses of vaccine typically required for the induction ofprotective immunity with protein subunit vaccines (e.g. hepatitis Bvirus formulated in alum adjuvant as described in Francis et al.; Ann.Int. Med. 97:362–6 (1982). Two additional breakthrough infectionsoccurred in vaccinees who had weak or undetectable primary (C7) andbooster (C15) responses. Of the three individuals who became infectedwith HIV-1 after receiving three or more productive immunizations (C10,C11, and C17), at least two, and possibly all three, appear to havebecome infected more than six months after receiving their lastimmunization. Because antibody titers to MN-rgp120 typically decay witha half-time of 2 to 2.5 months [Belshe et al.; JAMA 272(6):475–80(1994); Berman et al.; AIDS 8:591–601 (1994)], antibody titers would beexpected to have decayed at least eight-fold and possibly as much assixty four-fold at the time of infection. Thus, the lack of a sufficientimmune response at the time of infection represents a potentialexplanation for at least six of the seven breakthrough infections.

Data from vaccine efficacy studies in gp160 immunized chimpanzees[McElrath et al.; Longitudinal Vaccine-Induced Immunity and RiskBehavior of Study Participants in AVEG Phase II Protocol 201. In:Abstracts from Eighth Annual Meeting of the National Cooperative VaccineDevelopment Groups for AIDS. Bethseda, Md. 1996:216] challenged withHIV-1, and gp120-immunized rhesus macaques challenged with a chimericSIV/HIV-1 virus (SHIV) suggest that the magnitude of the neutralizingantibody response at the time of infection is a critical correlate ofprotective immunity. If maintaining neutralizing antibody titers provesto be a valid correlate of protective immunity in humans, thenformulations (e.g. novel adjuvants) or immunization regimes (frequentboosting) designed to maximize the antibody responses may be required toachieve long lasting protection. Use of a booster every six months maybe advantageous.

The other likely explanation for the late infections is the antigenicdifference between the vaccine and the breakthrough virus envelopeglycoproteins. This explanation is supported by the observation thatfour of the seven breakthrough viruses possessed envelope glycoproteinsthat differed from the MN-rgp120 by 25–30% at the amino acid level.Differences of this magnitude have historically [Myers et al.;Retroviruses and AIDS Database, Los Alamos National Laboratory (1992 and1995)] been associated with inter-subtype variation and far exceeds theaverage 10–20% variation expected for viruses within the same subtype.

Although the biologic significance of sequence variation in many regionsof the envelope glycoprotein is unclear, polymorphism at neutralizingepitopes is an important factor that affects vaccine efficacy. Previousstudies [Salmon-Ceron et al.; AIDS Res. and Human Retroviruses11:1479–86 (1996); Javaherian et al.; Science 250:1590—3 (1990)] havedemonstrated that the breadth of neutralizing activity that could beelicited by HIV-1 envelope derived vaccines was critically dependent onthe sequence of epitopes in the V3 domain (e.g.; the PND). Thus,candidate vaccines based on the LAI strain of HIV-1 (the prototypic“non-MN-like” subtype B virus), exhibited little or no crossneutralizing activity with subtype B viruses, whereas vaccines thatcontained the “MN-like-” PND sequence (IGPGRAF) (SEQ. ID. NO: 52)exhibited broad cross neutralizing activity. That four of the sevenbreakthrough viruses possessed envelope glycoproteins with radical aminoacid substitutions in the PND is consistent with the explanation thatdifferences in antigenic structure explain some of these infections.

Over the last few years, it has become clear that polymorphism among“MN-like” viruses occurs at neutralizing epitopes outside of the PND.The best example occurs in the C4 domain where two antigenicallydistinct variants are distinguished by the presence of either K or E atposition 429 [Moore et al.; AIDS 3:155–63 (1989)]. Because six of theseven breakthrough viruses differed from the vaccine strain in that theycontained E rather than K at position 429, antibodies raised to the C4domain of MN-rgp120 were unlikely to neutralize the viruses infecting insix of the seven vaccinees.

Other neutralizing epitopes are known to be present in the V1 and V2domains of gp120. Although these regions are highly variable, due toinsertions and deletions, neutralizing epitopes have been described byMcKeating et al.; J. Virol. 67:4932–44 (1993); Moore et al.; J. Virol.67:6136–6151 (1993); and Davis et al.; J. Gen. Virol. 74:2609–17 (1993).Several of these epitopes overlap an amino terminal sequence of the V2domain containing the tri-peptide sequence RDK at positionscorresponding to 142 to 144 of MN-rgp120 [McKeating et al.; J. Virol.67:4932–44 (1993); Moore et al.; J. Virol. 67:6136–6151 (1993)]. Likethe C4 epitope, variation in this sequence is known to occur betweendifferent substrains derived from the same parental isolate. Since allseven breakthrough viruses differed from MN-rgp120 in that theypossessed the RDK sequence, rather than the GDK sequence present in thevaccine antigen, neutralizing antibodies to the V2 domain of MN-rgp120would not have been expected neutralize any of the viruses recoveredfrom the vaccinees immunized with MN-rgp120.

Although polymorphisms at neutralizing epitopes might account for thelack of protection in most of the infections, this does not appear toexplain the infection of vaccinee C17, who was infected by a virus thatmatched MN-rgp120 in the V3 and C4 domains. If a difference in sequencewas responsible for the lack of protection in this case, the criticaldifference might relate to the unusual sequence in the V1 domain ofgp120 from this breakthrough virus. Several studies have shown that theV1 domain possesses epitopes recognized by virus neutralizing monoclonalantibodies [McKeating et al.; J. Virol. 67:4932–44 (1993); Davis et al.;J. Gen. Virol. 74:2609–17 (1993); Kayman et al.; J. Virol. 68:400–410(1994)].

Although far less is known about the V1 epitopes relative to otherneutralizing sites, the V1 epitopes appear to be conformation-dependent,and antisera from HIV-1 infected individuals recognize epitopes in theV1 and V2 domains [McKeating et al.; J. Virol. 67:4932–44 (1993); Kaymanet al.; J. Virol. 68:400–410 (1994)]. The V1 sequence of the virus fromC17 is noteworthy because it is smaller and contains fewer N-linkedglycosylation sites than that of MN-rgp120 or any of the otherbreakthrough viruses. By the same token, the envelope glycoproteins fromC11 and C6 are noteworthy because they are significantly larger andcontain more glycosylation sites than MN-rgp120 or the otherbreakthrough viruses.

While differences in amino acid sequence can provide clues todifferences in antigenic structure, the consequences of suchpolymorphism can only be proven through antibody binding studies. Tocorrelate differences in sequence with differences in antigenicstructure, gp120 from two clones each of all seven breakthrough viruseswas expressed and the antigenicity of the clones with a panel ofmonoclonal antibodies was examined. As predicted from the sequence data,none of the breakthrough virus envelope glycoproteins reacted withneutralizing MAbs to the V2 domain of MN-rgp120. When MAbs to the C4domain were examined, only the C17 envelope glycoprotein (that matchedMN-rgp120 with respect to K429) showed significant, albeit lower,binding. Surprisingly, the three breakthrough envelope glycoproteinsthat contained the subtype B PND consensus sequence, IGPGRAF (SEQ. ID.NO: 52), gave poor reactivity with all three PND directed MAbs, eventhough they possessed PND sequences closely related to the vaccineimmunogen. Thus, all three of the vaccinee isolates appeared to possesschanges outside of the recognition site that interfered with MAbbinding.

It has been known for many years that resistance to neutralization invitro can sometimes be attributed to mutations in remote sequences thatalter the conformation of neutralizing epitopes and interfere withrecognition by virus neutralizing antibodies [Nara et al.; J. Virol.64:3779–91 (1990); Cordonnier et al.; Nature 340:571–4 (1989)].Together, these results indicate that the antigenic structure of theenvelope glycoproteins recovered from the breakthrough viruses differedsignificantly from that of the vaccine antigen.

A novel result was the localization of residues in the C3 domain thatappeared to affect the binding of the virus neutralizing human MAb, 15e.This MAb is known to recognize a discontinuous epitope, block CD4binding, and neutralize a variety of laboratory and primary isolates ofHIV-1 [Ho et al.; J. Virol. 65:489–93 (1991); Thali et al.; J. Virol.66:5635–5641 (1992); Moore et al.; AIDS Res. Hum. Retroviruses9:1179–1187 (1993)].

Comparative binding to envelope glycoproteins from the breakthroughviruses indicated that recognition by this antibody is criticallydependent on residues in the C3 or C4 domains of gp120. The uniqueoccurrence of a positively charged K at position 351 in the C3 domainprovides a common explanation for the inability of the C11.5, C11.7 andC6.1 strains of HIV-1 to bind to 15e. Alternatively, it is possible thatdifferent amino acid substitutions in different locations account forthe failure of 15e to bind to rgp120s from the C6 and C11 clones. Theonly obvious positions where substitutions of this type occur are in theC4 domain where T replaces M at 434 (C11) and T replaces I at 439.

The present studies demonstrate that the current formulation ofMN-rgp120 is less than 100% effective against HIV-1 infection. Based onprevious in vitro and in vivo studies with MN-rgp120, protection fromnatural HIV-1 infection in humans is expected to depend on a thresholdconcentration of virus-neutralizing antibodies, and antigenic similaritybetween the vaccine immunogen and the challenge virus.

In this regard, only one of the seven breakthrough infections (C17) wasunexpected. This individual received a full course of immunizations yetbecame infected with a virus similar to MN-rgp120 at least two importantneutralizing epitopes (V3 and C4 domains). This infection might berelated to the magnitude of the antibody response at the time ofinfection, or antigenic differences between the breakthrough virus andthe vaccine strain, or circumstances of infection (e.g., ulcerativelesions, infection by donor with acute infection or high viremia), notmonitored in this protocol. Alternatively this individual may representa true vaccine failure, without clear explanation.

On balance, the analysis of breakthrough infections described herein didnot uncover any data that would discourage the continued development ofMN-rgp120 as a vaccine to prevent HIV-1 infection. The results supportspeculation that enhancing vaccine immunogenicity (as by additionalbooster immunizations) may be required to maintain long term protectiveimmunity, and that the addition of rgp120 from other antigenicallydifferent strains of virus in addition to MN-rgp120 are useful to expandthe breadth of protection.

The availability of viruses and viral glycoproteins derived frombreakthrough infections may provide an important means to streamline theprocess of identifying new antigens for inclusion into a multivalentvaccine. Recombinant viral glycoproteins prepared from breakthroughviruses, by definition, possess antigenic structures that aresignificantly different from MN-rgp120, and are be representative ofviruses currently being transmitted. Thus, combining rgp120 frombreakthrough viruses with MN-rgp120 is an effective way complement andsignificantly expand antigenic complexity and increase breadth of crossneutralizing activity.

1. A composition comprising one or more oligonucleotides capable ofexpressing a first polypeptide comprising a gp120 MN sequence asidentified by Sequence ID No. 41, or a fragment thereof, and apolypeptide comprising a breakthrough isolate gp120 sequence selectedfrom the group consisting of Sequence ID Nos. 2, 5, 8, 10, 12, 16, 19,23, 25, 28, 31, 33, 36, 39, and fragments thereof, in a suitablecarrier, wherein each of said fragments comprises at least the V2, V3,and C4 domains of gp120.
 2. The composition of claim 1 wherein a singleoligonucleotide expresses the first polypeptide and the polypeptidecomprising a breakthrough isolate gp120 sequence or a fragment thereof.3. The composition of claim 1 wherein said oligonucleotide or at leastone of said oligonucleotides is a DNA molecule.
 4. The composition ofclaim 1 wherein said oligonucleotide or at least one of saidoligonucleotides is a viral vector.
 5. A composition comprising: a) anoligonucleotide capable of expressing a first polypeptide comprising afirst gp120 sequence or a fragment thereof; and b) an oligonucleotidecapable of expressing a polypeptide comprising a breakthrough isolategp120 sequence, or a fragment thereof, wherein said breakthrough isolategp120 sequence is selected from the group consisting of Sequence ID Nos.2, 5, 8, 10, 12, 16, 19, 23, 25, 28, 31, 33, 36, and 39; wherein each ofsaid fragments comprises at least the V2, V3, and C4 domains of gp120,and said oligonucleotides are in a suitable carrier.
 6. The compositionof claim 5 wherein a single oligonucleotide expresses the firstpolypeptide and the polypeptide comprising a breakthrough isolate gp120sequence or a fragment thereof.
 7. The composition of claim 5 whereinsaid first gp120 sequence comprises gp120 MN as identified by SequenceID No. 41, gp120 CM244, gp120 MN-GNE6 which comprises Sequence ID Nos.43 and 44, or gp120 MN-GNE8 as identified by Sequence ID No.
 46. 8. Thecomposition of claim 5 wherein said composition additionally comprisesan oligonucleotide capable of expressing a second polypeptide comprisinga second gp120 sequence comprising gp120 MN as identified by Sequence IDNo. 41, gp120 CM244, gp120 MN-GNE6 which comprises Sequence ID Nos. 43and 44, gp120 MN-GNE8 as identified by Sequence ID No. 46, or a fragmentthereof, wherein said second gp120 sequence is different from said firstgp120 sequence.
 9. The composition of claim 8 wherein said first gp120sequence comprises gp120 MN as identified by Sequence ID No. 41 and saidsecond gp120 sequence comprises gp120 CM244.
 10. The composition ofclaim 8 wherein said first gp120 sequence comprises gp120 MN asidentified by Sequence ID No. 41 and said second gp120 sequencecomprises gp120 MN-GNE8 as identified by Sequence ID No.
 46. 11. Thecomposition of claim 8 wherein said breakthrough isolate gp120 sequenceor fragment is from a breakthrough isolate obtained from an individualimmunized with said first and second polypeptides.
 12. The compositionof claim 5 wherein at least one of said oligonucleotides is a DNAmolecule.
 13. The composition of claim 5 wherein at least one of saidoligonucleotides is a viral vector.
 14. A method for making acomposition comprising: a) providing a first composition comprising anoligonucleotide capable of expressing a first polypeptide comprising afirst gp120 sequence or a fragment thereof; b) obtaining a breakthroughisolate from an individual immunized with said first polypeptide; c)selecting a breakthrough isolate gp120 sequence, or a fragment thereof,from said breakthrough isolate; and d) adding an oligonucleotide capableof expressing a polypeptide comprising said breakthrough isolate gp120sequence, or fragment thereof, to said first composition; wherein eachof said fragments comprises at least the V2, V3, and C4 domains ofgp120.
 15. The method of claim 14 wherein said first gp120 sequence isfrom a macrophage-tropic HIV-1 strain.
 16. The method of claim 14wherein said first gp120 sequence is from a T-cell-tropic HIV-1 strain.17. The method of claim 14 wherein said first composition additionallycomprises an oligonucleotide capable of expressing a second polypeptidecomprising a second gp120 sequence, or a fragment thereof, from amacrophage-tropic HIV-1 strain.
 18. The method of claim 17 wherein saidfirst and second gp120 sequences bind to different chemokine receptors.19. The method of claim 18 wherein said first gp120 sequence binds toCC-CKR-5, and said second gp120 sequence binds to CXC-CKR-4.
 20. Themethod of claim 14 where said composition additionally comprises a virusengineered to induce a cytotoxic T-cell response.
 21. The method ofclaim 14 wherein said oligonucleotides are DNA molecules.
 22. The methodof claim 14 wherein said oligonucleotides are viral vectors.